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Department of Anatomy and Neurobiology (W.-W.L., A.Z.M., G.E.H.), University of Maryland, Baltimore, Maryland 21201; Department of Physiology (P.M.W.), University of Kentucky, Lexington, Kentucky 40536; and Department of Cell Biology (L.C.), Neurobiology and Anatomy, University of Cincinnati, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Dr. G. E. Hoffman, Department of Anatomy and Neurobiology, HSF 222, University of Maryland, School of Medicine, 685 West Baltimore Street, Baltimore, Maryland 21201. E-mail: gehoffma{at}umaryland.edu
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Abstract |
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Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r2 = 0.66; P < 0.01), suggesting that changes in the AVPv’s drive to LHRH neurons underlie the decrease in LHRH activity in middle age.
A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.
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Introduction |
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Neurons of the medial-most anteroventral periventricular preoptic area (AVPv) play a critical role in the stimulation of LHRH neurons at the time of the LH surge. The AVPv has had a number of names, making the integration of earlier studies difficult. Bleier (13) initially termed the area the periventricular preoptic area; and our initial studies of this area used the variant pePOA to designate the region (14, 15, 16, 17). Studies from Terasawa’s group (18, 19, 20) had termed this area the medial preoptic nucleus; but currently, a more caudal preoptic nucleus has adopted that name. Irrespective of its name, this region is located along the rostral half of the preoptic area along the ventricular surface in a strip of tissue approximately 70-µm wide and 300- to 400-µm long. The neurons of this medial AVPv innervate the LHRH neurons (21, 22) and are coordinately activated with LHRH neurons at the time of LH surges (14, 15). Lesions of the AVPv with no concomitant loss of LHRH neurons or their axons in the median eminence (23) block both spontaneous LH surges and surges evoked by exogenous E and progesterone (P) in ovariectomized rats (18, 19, 20, 24). These features suggest that a decline in afferent drive from AVPv neurons may account for the attenuation of LHRH activation in middle-aged rats. In the present study, we designed experiments to determine whether changes in the degree of Fos activation within the medial AVPv correlated with changes in LHRH neurons in young and middle-aged rats. A second set of experiments assessed whether elimination of the input from the medial AVPv neurons could mimic the reductions in LHRH Fos activation and LH secretion seen in middle age.
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Materials and Methods |
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Adult female Sprague Dawley rats that were young (3–4 months of age) or middle-aged (10–12 months) were housed under standard laboratory conditions on a 14-h light, 10-h dark schedule. One group of rats (n = 8 young, and n = 8 middle-aged rats) was ovariectomized and, 1 wk later, administered 17ß E2 at 5 h after lights on (180 µg/ml in sesame oil placed into SILASTIC brand capsules (Dow Corning, Midland, MI), 30 mm in length for young or 40 mm for middle-aged rats; capsules had an inner diameter of 0.062 inches and an outer diameter of 0.125 inches). Two days later, P (1.5 mg in 0.2 ml sesame oil) was injected sc to induce an LH surge. Rats were killed on the day of the expected LH surge, either near the peak of the LH surge (11 h after lights on; n = 4) or during the descending limb of the surge (13 h after lights on; n = 4). To determine whether the relationship extended to animals with spontaneous LH surges, we reanalyzed the AVPv and LHRH cells from sections generated in our earlier study (9). This group of rats (n = 5 young; n = 4 middle-aged) was monitored with daily vaginal smears to establish regular estrous cyclicity. Each rat chosen for study demonstrated consecutive 4-d cycles. Rats were killed on proestrus at the same times indicated for the E2 plus P (E+P) groups (11 and 13 h after lights on).
To assess the role of the medial AVPv in LH surge generation and activation of LHRH neurons, 49 ovariectomized young adult rats were anesthetized with chloral hydrate and placed into a stereotaxic apparatus, and 100–200 nl of a 3% solution of ibotenic acid (Sigma, St. Louis, MO; n = 41) or an electrolytic lesion (250–300 µA, 60 sec, n = 5) was directed toward the medial-most AVPv. After a 1- to 2-wk recovery, each animal received an sc injection of 5 µg E, 5 h after lights on. Twenty-four hours later, the rat received 50 µg E (Sigma, in 0.2 ml sesame oil, sc). The next day, at 6 h after lights on, each rat received 5 mg P (0.2 ml in sesame oil, sc). This protocol gives identical timing and amplitudes of LH surges as are found using SILASTIC-E capsules. All animals were killed on the day of P treatment, between 11 and 12 h after lights on (at the time of the induced LH surge in controls).
Preparation of tissue for histology
Ten to 15 min before death, all animals were anesthetized with
an overdose of pentobarbital (100 mg/kg), treated with heparin (1000
U), and perfused transcardially with saline containing 2% sodium
nitrite followed by 4% paraformaldehyde containing 2.5% acrolein
(25, 26). After a second saline flush, each animal’s
brain was removed, sunk in aqueous 25% sucrose, and sectioned on a
freezing microtome at 25 µm. Twelve sets of serial sections were
stored in cryoprotectant antifreeze solution (27) until
immunocytochemistry for Fos and LHRH was initiated.
Immunocytochemistry of Fos and LHRH
Immunocytochemical procedures were conducted as described
previously (25, 28, 29, 30, 31, 32, 33). Briefly, sections were rinsed
free of the cryoprotectant antifreeze solution, in potassium PBS (KPBS,
0.05 M, pH 7.4), reacted with 1% sodium borohydride in
KPBS, rinsed, and incubated for 48 h at 4 C in anti-Fos serum from
Dr. Philip Larsen (Panum Institute, Denmark) (serum 94012,
1:100,000), Oncogene Science, Inc. (Cambridge, MA)
(Ab 4191, 1:400,000), or from Cambridge Research Laboratories (Valley
Stream, NY; Ab 0A11-821, 1:40,000). All gave identical results. The
diluent for the serum contained 0.3% Triton X-100 in KPBS. The
sections were rinsed and incubated in biotinylated secondary
antibodies, either antirabbit (when used with antibodies from Dr.
Larsen and Oncogene Science, Inc.) or antisheep (when used
with antibody from Cambridge Research Laboratories) IgG (heavy
and light chains, 1:600 in PBS-Triton-X 100; Vector Laboratories, Inc., Burlingame, CA) for 1 h at room temperature.
Sections were again rinsed and incubated for 1 h at room
temperature in avidin-biotin complex (45 µl each of A and B Elite
reagents/ml KPBS-Triton-X 100; Vector Laboratories, Inc.).
After rinses in KPBS and then 0.175 M sodium acetate
solution, the sections were stained in a nickel sulfate (25
mg/ml)diaminobenzidine-HCl (DAB, 0.2 mg/ml) in sodium acetate solution
containing 0.83 µl 3%
H2O2, rinsed in acetate
solution, and transferred to KPBS. The sequence of reactions was then
repeated, substituting the primary antibody for LHRH. The LHRH
antiserum was a polyclonal antibody (gift of Drs. Robert Benoit and
Roger Guillemin, Salk Institute, La Jolla, CA), used at a
concentration of 1:100,000. The tissue was incubated in antirabbit IgG
antiserum (heavy and light chains; Vector Laboratories, Inc. 1:600) rinsed, and reacted with the avidin biotin complex
described above. The staining of the peroxidase reaction for LHRH was
accomplished with a standard diaminobenzidine solution to yield a brown
stain. The staining of Fos is carried to completion by visually
monitoring the staining reaction until no further labeling is observed
(approximately 15 min). In so doing, the deposited nickel DAB
prevents any possibility of erroneous labeling of unstained Fos when
the LHRH staining series is run. Fos is entirely nuclear and is never
found in the cytoplasm. Moreover, LHRH staining is always found in the
cytoplasm. When staining is properly monitored, no nuclear-only brown
staining is observed that would confound the results. Preabsorption of
the anti-Fos antibody with synthetic Fos (10 µg/ml diluted serum;
Peninsula Laboratories Inc., Belmont, CA) yields no
nuclear Fos staining. Preabsorption of the LHRH antibody with synthetic
LHRH (Abbott Laboratories, Abbott Park, IL; 0.5–1
µg/ml diluted serum) similarly blocks all staining of LHRH-containing
structures.
In animals that received lesions, one series of sections was stained for neuron nuclear antigen (NeuN) using a mouse monoclonal antibody (Chemicon) at a concentration of 1:50,000. Standard immunoperoxidase procedures, using nickel DAB as the chromogen (as described above), were followed.
Data analysis
AVPv/Fos. Sections from a 1-in-12 series, from each animal
stained with the double peroxidase method, were coded and counted by an
observer who was blind to the treatment of the animals. Three sections
from each animal, spaced 150 microns apart within the AVPv extending
from within 50 µm caudal to the organum vasculosum of the lamina
terminalis (OVLT) and extending caudally approximately 400 µm, were
chosen for analysis. Using a Nikon (Columbia, MD)
Optiphot microscope and a 10x objective (with a 10x eyepiece), the
observer scored each Fos-positive cell within a distance 70 µm from
the ventricular surface (determined by using an eyepiece grid). This
region was chosen for analysis because earlier studies determined that
Fos induction at the time of an LH surge was restricted to that narrow
AVPv zone (14). More lateral regions within AVPv were not
counted because they do not show any changes in Fos expression at the
time of the LH surge; this was also true of other subnuclei within the
preoptic area. Generally, the areas outside of the medial AVPv show
minimal Fos activation at any time during the cycle. Representative
rostral-caudal levels through this region are illustrated in Fig. 1
. The boxes depicted on
the right side of the figure show the number, positions, and size
of grid boxes in which Fos+ nuclei were counted. A Mann-Whitney
U test was used to compare values from the young and old
groups; differences were considered significant at the level of
P 
0.05.
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0.05. Correlation between AVPv and LHRH Fos activation. The relationship between the number of medial AVPv Fos+ neurons and the proportion of LHRH neurons that expressed Fos was determined with a Spearman Rho nonparametric correlation analysis; P < 0.05 was considered significant.
Analysis of lesion data. Initial histological examination of sections stained with NeuN was used to determine which animals had AVPv lesions. Next, patterns of Fos activation in LHRH neurons from animals with lesioned medial AVPv were compared with those in which the medial AVPv was intact. To determine whether lesions of the medial AVPv reduced secretion of LH, the animals were divided into three groups based on the integrity of the medial AVPv: 1) animals with no detectable lesion in the brain (n = 21) and animals with missed lesions (n = 21); 2) animals with partial medial AVPv lesions (n = 3); and 3) animals with complete unilateral lesions of the medial AVPv (n = 4). Plasma LH values were compared using nonparametric Mann-Whitney U tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the increase in Fos activation in
LHRH neurons seen in young animals was significantly reduced in the
middle-aged animals (23.8% ± 2.7 for the E+P group, and 20.8% ± 9.5
for cycling rats). On average, Fos activation in LHRH neurons
accompanying the LH surge in young rats was just over twice that
observed in middle-aged animals, irrespective of the manner in which
the LH surge was generated. No differences between animals killed at
the peak of LH secretion and those killed during the early phase of the
descending limb of the LH surge was noted. Within AVPv (Fig. 2B), Fos
activation in the middle-aged animals was also greatly reduced at the
time of the LH surge (from 296.8 ± 40.4 to 116.3 ± 40.8 for
proestrous rats and from 270.8 ± 23.0 to 132.1 ± 15.8 for
ovariectomized steroid-replaced rats). Comparisons between the
proportion of LHRH neurons that expressed Fos and the number of Fos+
medial AVPv neurons for both young and middle-aged animals revealed a
striking positive correlation (Fig. 3, r2 = 0.66; P = 0.01).
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shows
2 animals with ibotenic acid lesions that eliminated the majority of
neurons in the rostral preoptic area on 1 side of the brain. In one
(Fig. 4A), the medial AVPv was eliminated; in the other (Fig. 4B), it
was spared. In the animal whose medial AVPv was destroyed, AVPv Fos
activation was noted only on the intact side (Fig. 4C), and LHRH Fos
activation was blocked on the lesioned side (Fig. 4E). In the other
animal, Fos activation in the medial AVPv was quite normal (Fig. 4D),
and LHRH neurons were strongly activated on both sides of the brain
(Fig. 4F). One of the animals that received an electrolytic lesion had
a bilateral lesion of the medial AVPv that extended only 100 µm from
the ventricular surface (Fig. 5A). In
that animal, LHRH neurons failed to activate on either side of the
brain (Fig. 5, B and C). Fig. 6 shows a
summary of the plasma LH values for the animals in the lesion study. As
more and more of the medial AVPv was eliminated, LH levels at the time
of the expected LH surge declined; and when 1 side of the medial AVPv
was completely destroyed, plasma LH was significantly lower than that
of animals with no detected lesion or with lesions that missed the
medial AVPv. The animals bearing partial medial AVPv lesions were
intermediate. One of the electrolytically lesioned rats had a lesion
placement that shifted rostrally and eliminated the majority of LHRH
neurons. That animal had a plasma LH value that was lower than that in
the animals with unilateral AVPv lesions and was similar to that in
animals that had no LH surge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies from our laboratories, as well as those of others, indicated that the reduction in the number of LHRH neurons expressing Fos in middle-aged rats predicted the decline in peak LH values at the time of an LH surge (7, 9). The present data extend these findings and demonstrate that parallel reductions in the activation of neurons in the AVPv accompany the decline in LHRH activation that occurs during middle age. The reduction of Fos activation within both AVPv and LHRH neurons suggests that the two events are linked, either through parallel common afferents that regulate both LHRH and AVPv neuronal activity or through a sequential series of neurochemical signals. Because the AVPv neurons directly innervate LHRH neurons (15, 21), this suggests that a deficit in AVPv stimulation determines the reduction in LHRH activation. This conclusion is supported by earlier studies showing that electrolytic lesions of the entire AVPv block the LH surge (18, 19, 20, 24). Our present data (which indicate that when ibotenic acid or electrolytic lesions destroy the medial AVPv, steroid treatment fails to evoke LHRH activation on the side ipsilateral to the lesion, and LH secretion is reduced) support this hypothesis. Interestingly, lesions placed more laterally or rostrally that exclude the medial AVPv had no effect on LHRH Fos induction, further suggesting that medial AVPv is the central component. The fact that, in middle-aged rats, the LHRH neurons remain responsive to potassium stimulation to the same extent as cells from younger rats (37), further indicates that it is stimulatory drive to LHRH neurons that is reduced in aging.
What produces the reduced drive to the AVPv neurons during middle age is unknown. One possibility, based on the initial studies of cycling animals, was that E was either not secreted in sufficient amounts to trigger proper up-regulation of PRs, or that the P secretion was deficient. However, the studies of Wise and Lapolt et al. (38, 39) show that E and ER levels are maintained or elevated in middle-aged (compared with young) rats. We assayed tissue from young and middle-aged rats and found that binding of labeled P (39) and PR protein patterns (G. E. Hoffman, unpublished observation) were indistinguishable in the young and middle-aged preoptic areas. The fact that the deficit was detectable in middle-aged rats that were still regularly cycling, as well as in ovariectomized rats that received controlled concentrations of E and P, further indicates that decline in steroid levels is not the cause of reproductive failure. More likely, the declining responsiveness of AVPv neurons to other inputs is involved. One of the extrinsic factors needed for LH surges is the diurnal elevation of cAMP levels in the AVPv (40). Indeed, our preliminary data suggest that this diurnal rhythm in cAMP levels is no longer detectable in middle-aged rats (P. M. Wise, unpublished observation). Such a loss in available cAMP may render the AVPv neurons less responsive to P and prevent their ability to stimulate LHRH neurons.
Age-related alterations in activation of neurons in the AVPv are likely to have major repercussions on LHRH secretion, because an accumulating body of evidence demonstrates that the AVPv is a critical nodal relay station that integrates a host of hormonal and neuronal inputs that regulate LHRH secretion. Catecholamines, serotonin, and numerous peptide afferents terminate in this region (41). Moreover, AVPv cells contain both ER and PR (22, 42, 43). Use of either hormonal paradigms associated with surge blockade (14) or administration of transmitter receptor blockers that block LH surges (34) (G. E. Hoffman, unpublished data) block or attenuate Fos expression in the medial AVPv in parallel with blockade of LHRH Fos activation. Earlier studies, which used lesions of the entire AVPv, determined that elimination of the AVPv blocked LH surges (18, 19, 20, 24). Our data further indicate that it was the medial AVPv that produced that effect. Together, these data suggest that the AVPv functions as an integrative center for the regulation of LHRH neuron firing and that disruption of any of inputs either hormonal or neuronal, alters the drive these neurons impose on the LHRH system.
In summary, our findings demonstrate that changes in Fos activation in neurons in the AVPv occur in middle-aged proestrous and steroid-treated rats in parallel with changes in Fos activation within LHRH neurons. Lesions of the medial AVPv produce reduced LHRH activation and LH secretion. These data strongly suggest that the changes in Fos activation, during middle age, reflect altered stimulatory activity in the AVPv neurons responsible for the attenuated activity in LHRH neurons, leading to a decline in the amplitude and a delay in the onset of the LH surge that we observed previously.
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Acknowledgments |
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Footnotes |
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Abbreviations: AVPv, Anteroventral periventricular preoptic area; DAB, diaminobenzidine HCl; E+P group, group that was administered E2 plus P; KPBS, potassium PBS; NeuN, neuron nuclear antigen; OVLT, organum vasculosum of the lamina terminalis; P, progesterone.
Received December 27, 2000.
Accepted for publication July 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-1
Adrenergic receptor blockade blocks LH secretion but not LHRH cFos
activation. Brain Res 747:236–245[CrossRef][Medline]
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