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Division of Neuroscience, Oregon Regional Primate Research Center, Department of Physiology and Pharmacology, Oregon Health Sciences University, Beaverton, Oregon 97006
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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Introduction |
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Another important unanswered question regarding the possible effects of NPY on GnRH neurons is the receptor subtypes that may be important in mediating the potential effects of NPY. Pharmacological studies have suggested that NPY Y1 receptors are involved in the central effect of NPY in mediating LH secretion (12, 13). In addition, Y1 receptor is expressed in the GnRH-like tumor cell line, GT1–1 cells (14). Thus, these results suggest that Y1 receptors may be expressed in endogenous GnRH neurons to mediate the effects of NPY.
To provide an anatomical basis for the potential direct effect of ARH NPY on GnRH neurons, morphological studies were conducted in lactating rats. This animal model was chosen because several studies have shown that the activity of the ARH NPY neurons is significantly elevated during lactation (15, 16, 17), resulting in a significant increase in NPY content, thus making it easier to detect NPY fibers and nerve terminals. In addition, a reduced GnRH neuronal activity and/or secretion has been postulated to account, in part, for the suppression of LH secretion that is characteristic of lactation (18, 19, 20). One possible candidate that may be involved in the suppression of LH secretion is NPY. Furthermore, during lactation, NPY immunostaining in ME is greatly enhanced, compared with the nonlactating rat (21, 22), implying that during lactation, NPY input in the ME may have greater impact on GnRH secretion from its terminals. Thus, the possible direct interaction between the ARH NPY system and GnRH neurons has relevance to an understanding of the regulation of cyclic reproductive function during lactation.
The present studies first used the anterograde tracer, Phaseolus vulgaris leucoagglutinin, PHA-L (23), combined with multiple immunofluorescent staining, to examine whether, in lactating rats, NPY neuronal fibers from the ARH make close appositions to GnRH perikarya, as well as to its nerve terminals. In the second experiment, the presence of NPY Y1 receptor expression on GnRH neurons was examined by double-label immunostaining.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anterograde tracer injection
Lactating animals suckling eight pups received the tracer
injection on day 1 postpartum. Animals were anesthetized with
tribromoethanol (20 mg/100 g BW) and placed in a stereotaxic apparatus.
A glass micropipette with tip diameter of 10–20 µm was filled with
the anterograde tracer, PHA-L (Vector Laboratories, Inc.,
Burlingame , CA; 2.5% wt/vol, in filtered phosphate buffer),
and inserted into the region of the ARH. Injection coordinates were
3.0–3.3 mm caudal, 0.2 mm lateral to the bregma, and 9.35 mm below the
dura for the caudal portion of the ARH, according to the atlas of
Paxinos and Watson (24). The caudal portion of the ARH was chosen
because increased NPY activity is restricted to this area during
lactation 15, 17). PHA-L was injected by iontophoresis with 5 µA
current, pulsed at 7-sec intervals for 20 min. The glass pipette was
left in situ for an additional 5 min before retrieving.
After the injection, the animals were returned to their pups.
Perfusion and tissue sectioning
Ten to 12 days post injection, lactating animals were
anesthetized with an overdose of pentobarbital (125 mg/kg BW, ip) and
perfused transcardially with 150 ml of 2% sodium nitrite in saline,
followed by 350 ml 3.8% of borax in 4% paraformaldehyde (pH 9.5). The
brain was removed and immersed in 25% sucrose at 4 C for 6 h.
Coronal sections for the whole brain (25 µm) were cut on a sliding
microtome and collected in a one-in-four series. The tissue sections
were stored at -20 C in multiwell tissue culture plates containing
cryoprotectant until use.
Immunocytochemistry procedures
Verification of injection site. Tissue sections containing
the ARH were rinsed in 0.05 M potassium PBS (KPBS) several
times and incubated in rabbit anti-PHA-L antibody (1:500; DAKO Corp., Carpinteria, CA) in KPBS with 0.4% Triton X-100 at room
temperature for 1 h. After the incubation, the tissue was rinsed
with KPBS and incubated in fluorescein isothiocyanate-conjugated goat
antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA) for 1 h. After incubation, the sections were
rinsed, mounted on glass slides, air dried, and coverslipped with
buffered glycerol. The staining was examined under an epifluorescent
microscope. Five animals with injection sites centered in the ARH were
used in the following studies.
Multiple label immunofluorescence. For GnRH, NPY, and PHA-L triple labeling, the tissue sections were incubated in a mixture of rabbit anti-PHA-L antibody (1:1000), goat anti-NPY antiserum (1:4000, provided by Dr. Philip J. Larsen at Novo Nordisk A/S, Glostrup, Denmark), and the mouse anti-GnRH antibody (1:600, provided by Dr. Henryk Urbanski at the Oregon Regional Primate Research Center) in KPBS with 0.4% Triton X-100 and 5% nonfat milk at room temperature for 1 h, followed by 4 C for 48 h. After several washes with KPBS, the sections were incubated in a mixture of affinity purified secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.): donkey antirabbit IgG conjugated with biotin (1:600), donkey antigoat IgG conjugated with tetramethyl rhodamine (1:300), and donkey antimouse IgG conjugated with indodicarbocyanine (Cy5, 1:300) at room temperature for 1 h, followed by incubation in avidin-conjugated fluorescein isothiocyanate (1:1000) for 1 h. After the incubation, the sections were rinsed, mounted with buffered glycerol, and coverslipped.
For GnRH and NPY Y1 receptor double labeling, tissue sections were incubated in the antibody mixture containing rabbit anti-Y1 receptor antiserum (1:7000, provided by the CURE/Gastroenteric Biology Center, Antibody/RIA Core, UCLA, Los Angeles, CA) and mouse anti-GnRH antibody (1:1000). The specificity of the anti-Y1 receptor antiserum has been evaluated in earlier studies (25, 26). In the present studies, the specificity was further confirmed by preabsorbing the antibody with the Y1 receptor fragment used to generate the antibody. The secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) used were donkey antirabbit IgG conjugated with biotin (1:600) and donkey antimouse IgG conjugated with cy5 (1:400). Y1-immunoreactive signal was further amplified with tyramide signal amplification-indirect technology using a commercial kit (tyramide signal amplification-indirect kit; NEN Life Science Products, Boston, MA) and was visualized with Cy2-conjugated streptavidin (1:1000, Jackson ImmunoResearch Laboratories, Inc.).
Image analysis
The triple-labeling results were analyzed with the aid of
confocal laser scanning microscopy. The Leica Corp.
(Germany) TSC NT confocal system consisted of a Leica Corp. RBE inverted microscope equipped with a Ar laser producing
light at 467 nm and 488 nm, a Kr laser for 568 nm, and a HeNe laser for
647 nm light. Various objectives (25x/NA 0.75, 40x/NA 1.25, and
100x/NA 1.4) were used to scan the images. A series of continuous
optical sections, at 0.78-µm intervals along the z-axis of a tissue
section, were scanned for all 3 fluorescent signals. The signals
obtained for each fluorophore on one series of optical sections were
stored separately as a series of 512 x 512 pixel images. The
images were then processed with the MetaMorph Imaging System (Universal
Imaging Corporation, West Chester, PA). The confocal images are
presented as projections (a maximum value projection is a series
of 32 individual optical planes covering 25 µm, or as single optical
planes. PHA-L and NPY single-labeled images were first combined
together to obtain the NPY fibers that also stained positively for
PHA-L. The NPY/PHA-L double-labeled images were then combined with
GnRH-stained images to analyze for the presence of close appositions
between the NPY/PHA-L double-labeled fibers and GnRH-positive neurons
in POA and GnRH terminals in ME. Similar procedures were used for
analyzing the GnRH/Y1 double- labeling results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were
used for the multiple-labeling studies. When PHA-L fibers were examined
under dark field, dense tracer-labeled fibers were found mainly in the
hypothalamus, including the medial POA (mPOA), periventricular POA, the
parvocellular portion of the paraventricular nucleus, dorsomedial
nucleus, the ARH, and the ME (data not shown). Outside the
hypothalamus, PHA-L fibers were found mainly in the ventral part of the
lateral septum and the bed nucleus of the stria terminalis. It should
be pointed out that one PHA-L injection labels only a small number of
neurons surrounding the injection site (23). Thus, the PHA-L/NPY
double-labeled fibers observed in the present study represent only a
small percentage of NPY fibers from the ARH. Because only a small
portion of ARH NPY neurons are labeled after one PHA-L injection, it is
not possible to quantify the portion of NPY input to GnRH neurons that
is derived from the ARH.
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, a–c, NPY and PHA-L single-labeled
images were combined to identify NPY/PHA-L double-labeled fibers.
GnRH-positive neurons (Fig. 2d, green) were found scattered
in the OVLT and the rostral end of the POA. NPY single-labeled fibers
(white), as well as NPY/PHA-L double-labeled fibers
(orange), which represent the NPY fibers from the ARH NPY
neurons, were also found (Fig. 2). When multiple staining signals were
examined at higher magnification, NPY/PHA-L double-labeled fibers made
close appositions on GnRH-positive neurons in the OVLT area (Fig. 3). An examination of 0.78-µm single
optical sections through each GnRH neuron demonstrated that the
double-labeled fibers did make contact with the cell bodies. Although
these results did not permit an assessment of the portion of NPY input
to GnRH neurons that was derived from the ARH, some quantitative
evaluation could be made. In general, each 25-µm tissue section of
the OVLT/POA contained, on average, approximately 20 GnRH cell bodies,
and almost all cell bodies were surrounded by NPY fibers. Nearly 50%
of the GnRH cell bodies showed close appositions with NPY neurons, and
about one-fifth of those close appositions represented NPY neurons from
the ARH.
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Triple labeling for GnRH, NPY, and PHA-L in the ME
In the ME, procedures similar to those described above were used
to identify NPY and PHA-L single-labeled fibers, as well as NPY/PHA-L
double-labeled fibers. The GnRH-positive fibers and nerve terminals in
the ME were concentrated in the external layers, whereas NPY-positive
fibers were found mainly in the internal layers (Fig. 4). Nonetheless, many NPY-positive, as
well as NPY/PHA-L double-labeled fibers, were observed in the external
layers, which were in the vicinity of the GnRH fibers and nerve
terminals. About 30% of the NPY fibers were double labeled with PHA-L.
At higher magnification (Fig. 5),
numerous double-labeled fibers could be seen making very close
appositions (within 0.78 µm) on GnRH terminals.
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, control
peptide preabsorption completely eliminated Y1 staining from the brain
sections. The Y1 immunostaining pattern obtained was identical to
previously reported studies (25, 26) and included labeling of both cell
bodies and nerve fibers (data not shown). The GnRH and Y1 double-label
staining was examined with confocal microscopy. In the OVLT/POA area,
where the GnRH cell bodies are located, intensive Y1 receptor staining
was observed (Fig. 7). Most of the Y1
staining resembled neuronal fibers, but occasional cell body staining
was also found. When GnRH and Y1 staining were visualized
simultaneously, most GnRH cell bodies were found surrounded by
Y1-positive fiber staining, but no convincing colocalization between
GnRH-positive and Y1-positive cells could be found (Fig. 7). Some
Y1-positive fibers were found in close apposition to the GnRH cell
bodies (Fig. 7).
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) and ME (Fig. 9), where intensive GnRH neuronal fibers
and nerve terminals are located, strong Y1-positive fibers were also
observed. In addition, Y1 immunostaining was found in the vasculature
outside the brain (Figs. 8 and 9). When GnRH and Y1 staining in the ME
were viewed simultaneously under higher magnification, many Y1-positive
fibers were also GnRH-positive (Fig. 9, b–d). In addition, some close
appositions between GnRH and Y1-positive fibers were found in the same
area (Fig. 9, b–d).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, the present study also showed that ARH NPY fibers are present in the ME and make close appositions on GnRH fibers. This result suggests that ARH NPY input may modulate GnRH secretion from its nerve terminals in the ME. It has been shown that NPY suppresses LH secretion when the steroid hormone levels are low, such as during lactation (12), whereas under conditions when steroid hormone levels are elevated, such as during proestrus of the estrous cycle, NPY can stimulate GnRH secretion from the ME (12). Thus, it is reasonable to speculate, based on the morphological data provided by these studies, that the ARH NPY input to GnRH terminals in the ME may play a role in the regulation of GnRH secretion during different reproductive states when steroid hormone secretion is altered. However, more studies are needed to further elucidate this issue. In addition, it has been well established that NPY is released into the hypophysial portal vasculature (27, 28). The presence of ARH NPY in the ME, particular the external layer, suggests that the ARH is a source of NPY in the portal system. Thus, ARH NPY may also be having direct effects at the pituitary level, because NPY has been shown to modulate the secretion of several hormones from the anterior pituitary (12, 29, 30, 31, 32).
To begin to assess the receptor subtypes expressed on GnRH neurons that may mediate the effects of ARH NPY input, the present study demonstrated the expression of Y1 receptor on GnRH neurons, using an antibody specific to the Y1 receptor. The specificity of the antibody has been evaluated in several earlier studies (26, 27). In addition, the amino acid sequence to which the antibody was raised is specific to the Y1 receptor. Furthermore, in the present study, the complete absence of staining after the antibody was preabsorbed with the Y1 receptor fragment used to generate the antibody further confirmed that the antibody was specific to the Y1 receptor peptide. The present study showed that Y1 staining in GnRH neurons was predominantly in axons, rarely was it found in cell bodies. The lack of Y1 staining in GnRH cell bodies could not be attributed to the inability of the antibody to stain cell bodies, because numerous Y1-positive cell bodies were observed throughout the hypothalamus, including the POA. However, the demonstration of possible direct ARH NPY input to the GnRH neurons in the POA implies that there may be additional NPY receptors, other than Y1, expressed on the perikarya to mediate the effect of ARH NPY input.
Although no convincing Y1 staining was found in GnRH cell bodies, Y1-positive fibers made close appositions on GnRH cell bodies in the OVLT and rostral end of the POA. This observation suggests that in these areas, NPY may be able to indirectly modulate GnRH neuronal activity by synapsing on the Y1-positive fibers to modulate the secretion of neurotransmitters or modulators from the nerve terminals of the Y1-positive fibers. The released substrates would then regulate GnRH neuronal activity. This notion is supported by a recent study in which NPY was shown to modulate CRF neuronal activity in the paraventricular nucleus of the hypothalamus by a presynaptic mechanism to modulate GABA release from the presynaptic terminals (Roger D. Cone, personal communication).
The demonstration of Y1 receptor on GnRH axons suggests that Y1 receptor may function as a presynaptic receptor to mediate the possible effect of NPY on GnRH secretion from its nerve terminals. Several studies have demonstrated that the Y1 receptor in several brain areas (such as the suprachiasmatic nucleus, the nucleus accumbens, and the ARH) is expressed presynaptically (33, 34, 35), even though the Y1 receptor has been considered, in general, as a postsynaptic receptor (34, 36). Functionally, the activation of presynaptic Y1 receptors has been shown to be associated with the suppression of transmitter release primarily through inhibition of Ca2+ channels (37, 38, 39, 40). Thus, it is possible that during lactation, increased amounts of NPY in the ME may bind to presynaptic Y1 receptors to suppress GnRH secretion from its nerve terminals.
On the other hand, it has been shown that NPY can also stimulate GnRH release from ME fragments obtained from proestrus rats (41), and a study by Leupen et al. (13) suggested that the stimulatory effect of NPY may be mediated by the Y1 receptor. Thus, more studies are needed to further elucidate the function of Y1 receptors on GnRH nerve terminals under conditions when it is exposed to different patterns and levels of NPY in the ME. The present study does not rule out the involvement of other receptor subtypes in mediating the effect of NPY in the ME. In fact, a pharmacological study by Barker-Gibb et al. (42) suggested that Y2 receptor may also be involved in the presynaptic action of NPY on GnRH nerve terminals in the ewe.
In conclusion, the present study demonstrated that ARH NPY neurons come in close contact with GnRH neurons in the POA and with their nerve terminals in the ME. Therefore, ARH NPY neurons may directly regulate GnRH neuronal function. GnRH and NPY Y1 receptor double-label studies demonstrated that the Y1 receptor is expressed on GnRH fibers and nerve terminals but is rarely found in GnRH cell bodies. These results raise the possibility that Y1 receptor in the ME may mediate the effects of NPY to modulate GnRH secretion from nerve terminals.
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Acknowledgments |
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Footnotes |
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Received April 7, 1999.
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kanson R 1986 Evidence for different pre and post junctional receptors for
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