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Endocrinology Vol. 140, No. 1 510-519
Copyright © 1999 by The Endocrine Society

ARTICLES

Periventricular Preoptic Area Neurons Coactivated with Luteinizing Hormone (LH)-Releasing Hormone (LHRH) Neurons at the Time of the LH Surge Are LHRH Afferents1

Wei Wei Le, Kathie A. Berghorn, Stefanie Rassnick and Gloria E. Hoffman

Department of Anatomy and Neurobiology, University of Maryland School of Medicine (W.W.L., G.H.), Baltimore, Maryland 21201; the Laboratory for Pregnancy and Newborn Research, Cornell University (K.A.B.), Ithaca, New York 14853; and the Department of Neuroscience, University of Pittsburgh (S.R.), Pittsburgh, Pennsylvania 15217

Address all correspondence and requests for reprints to: Gloria E. Hoffman, Ph.D., Department of Anatomy and Neurobiology, University of Maryland, Room 222 HSF, 685 West Baltimore Street, Baltimore, Maryland 21201. E-mail: gehoffma{at}umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Earlier studies demonstrated coactivation of the periventricular preoptic area (pePOA) with LHRH neurons at the time of an induced or spontaneous LH surge, suggesting that the pePOA might regulate LHRH neurons. To investigate this hypothesis, studies were conducted to determine the temporal pattern of pePOA Fos activation during the rat estrous cycle and establish the connections of the pePOA neurons with LHRH neurons. Fos activation within LHRH and pePOA neurons showed the same temporal pattern. Both were absent during diestrous I, diestrous II, and the morning of proestrus. Fos was induced in the pePOA and LHRH neurons beginning on the afternoon of proestrus (4 h before lights off), with a decline 8 h later on proestrous evening. Tract-tracing studies then established the relationship between LHRH and pePOA neurons. Retrograde labeling with fluorogold determined that a portion of the Fos-positive pePOA neurons present at the time of the LH surge sent a projection to regions that contain LHRH cells. Anterograde tracer (neurobiotin) injections established that the pePOA neurons sent axons to the LHRH cells. Taken together, these data indicate that the pePOA provides direct input to LHRH neurons that is likely to stimulate LHRH neurons at the time of the LH surge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PERIVENTRICULAR zone of the preoptic area (pePOA) is one of a number of sexually dimorphic nuclear regions, with neuronal density greater in the female than in the male (1). Calculations of cell number for any of the phenotypically identified neurons found in that region [e.g. dopamine (2), atrial natriuretic peptide (3), cholecystokinin (4), and neurotensin (5)] show greater numbers of neurons in females than in males.

A number of sexually dimorphic functions, including sexual posturing (6, 7), maternal behavior (8, 9), and the ability to display cyclic LH surges (10, 11, 12), are associated with subdivisions of the preoptic area. Studies with the use of Fos as an activity marker in preoptic area neurons during sexual behavior (13, 14, 15, 16) have led to the conclusion that the medial preoptic nucleus, centrally located within the POA, is a part of the circuitry subserving reproductive behaviors. A similar role for the pePOA in these same behaviors has not been demonstrated.

Previous studies noted that after estrogen (17) or estrogen plus progesterone (16) treatment, neurons along the POA’s periventricular zone strongly expressed Fos-like proteins. The times that Fos proteins were induced coincided with the times LH surges would be evoked, suggesting that activation of the pePOA might be linked to stimulation of LHRH neurons. This feature is further supported by our recent studies demonstrating that immature animals treated with estrogen and progesterone so as to induce a LH surge had simultaneous activation of both LHRH and pePOA neurons; by contrast, animals treated with steroids in a paradigm where LH surges were inhibited failed to show either LHRH or pePOA Fos activation (18). That the pePOA might direct activity in LHRH neurons is further suggested by anatomical studies in which degenerating axon terminals synapsing onto LHRH neurons were observed after neurotoxin (6-hydroxydopamine) lesions of the preoptic area (19). As most of the dopamine neurons of the POA are located within the pePOA (2), these data raise the likelihood that the pePOA participates directly in LHRH control.

The literature cited above supports the idea that the pePOA actively participates in the LH surge mechanism, but fails to address the question of whether pePOA activation is synchronized to LHRH activation and whether there exist direct connections of activated pePOA neurons with LHRH neurons. To address these questions, we conducted two studies. One examined the temporal pattern of Fos induction in neurons of the pePOA in cycling rats and compared those results to LHRH cell activation patterns to test the hypothesis that the pePOA activation was synchronized to that of LHRH neurons. A second series of experiments used tract tracing to determine whether neurons retrogradely labeled from regions that contain LHRH neurons were activated at the time of the LHRH surge, and whether anterograde labeling of pePOA axons came into close contact with LHRH neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Sprague-Dawley rats purchased from Zivic-Miller Laboratories, Inc. (Zelienople, PA) were kept under standard laboratory conditions (12-h light, 12-h dark cycles, with lights on at 0400 h), with food and water available ad libitum. The University Committee on Animal Research approved all protocols. Daily vaginal smears performed at 1000 h determined the estrous cycle stage.

For the experiments examining the coordination of LHRH and pePOA neuronal Fos activation, 29 rats exhibiting 3 consecutive 4-day estrous cycles were chosen for further study. Animals were killed at various times during the estrous cycle, as indicated in Table 1Go, after being anesthetized with an overdose of pentobarbital (100 mg/kg). All animals were administered 1000 U heparin, and a blood sample was taken directly from the heart for determination of plasma LH values. Subsequently, each rat was perfused transcardially with saline containing 2% sodium nitrite followed by 2.5% acrolein in buffered 4% paraformaldehyde (20). All brains were sunk in 25% aqueous sucrose and sectioned on a Reichert AO freezing microtome (AO Instruments, Buffalo, NY) into a 1 in 12 series of 25-µm sections. Sections were stored in cryoprotectant (21) until staining for Fos and LHRH was initiated.


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  		Table 1. Relationship between LHRH and pePOA activation during the estrous cycle

 
For determining whether activated pePOA neurons could be retrogradely labeled from collections of LHRH neurons, a group of cycling rats (n = 15) was injected with the retrograde tracer fluorogold (FAu, Fluorochrome, Englewood, CO) placed into the POA at sites where LHRH neurons are most numerous [adjacent to the organum vasculosum of the lamina terminalis (OVLT) or within 400 µm caudal to the OVLT along the borders of the medial and lateral preoptic areas] (22). These rats were anesthetized with Equithesin anesthesia (0.33 ml/100 g body weight ip of a solution of sodium pentobarbital 0.98 g/dl, chloral hydrate 4.25 g/dl, and MgSO4 2.12 g/dl) and placed in a stereotaxic frame (David Kopf Instruments). Glass capillaries with a tip od of 20–40 µm were filled with 0.9% NaCl solution containing 4% FAu and lowered stereotaxically into LHRH cell-containing regions (23) aided by the Paxinos and Watson atlas (24). Iontophoretic injections were made into the POA using a constant positive current of approximately 3.9–4.0 mA through the capillary. Injection times were varied between 30 sec and 2 min to achieve a minimal degree of dye spread (<200 µm) into the injected area. Animals were allowed to recover for a minimum of 14 days, which is sufficient to allow retrograde transport of the dye to all cell bodies with terminals within the injection site. Two or 3 days after tracer application, vaginal smears were again monitored. After three consecutive cycles, animals were selected on proestrus at the time of expected peak LH secretion, anesthetized, and killed.

A second series of cycling female rats (n = 17) received stereotaxic injections of the anterograde tracer neurobiotin (Vector Laboratories, Inc., Burlingame, CA) placed into the pePOA. Glass capillaries with a tip of 20–40 µm od were filled with 0.9% NaCl solution containing 5% neurobiotin and lowered stereotaxically into the pePOA, 300–600 µm caudal from the OVLT [aided by use of a stereotaxic atlas (24)] under pentobarbital anesthesia. The capillaries were connected in a fashion identical to that used for FAu injection; injection times are varied between 5–10 min (with current delivered 10 sec on, 10 sec off) to achieve a minimal degree of dye spread (<100 µm) into the injected area. After a 24-h survival time, the animals were anesthetized and perfused with a 4% paraformaldehyde-2.5% acrolein solution. The brains were removed, sunk in 25% aqueous sucrose, and stored in cryoprotectant solution until they were stained for double labeling of LHRH and neurobiotin.

Immunocytochemistry
Fos and LHRH. Staining for Fos in combination with LHRH used essentially the same strategy as that previously reported (20, 25, 26, 27, 28, 29, 30, 31). Briefly, the sections (from a 1 in 12 series) for immunocytochemical staining were removed from the cryoprotectant, rinsed in PBS, treated with a 1% NaBH4 solution (Sigma Chemical Co., St. Louis, MO), rinsed, and then incubated with anti-Fos antibody (Oncogene Science, Inc., Tarzana, CA; AB-2; 1:50,000, or 0.02 µg/ml) in PBS with 0.4% Triton X-100 for 48 h at 4 C. After rinsing, the tissue was incubated for 1 h at room temperature in biotinylated goat antirabbit IgG (heavy and light chains; Vector Laboratories, Inc.) at a concentration of 1:600 in PBS with 0.4% Triton X-100, rinsed, and incubated for 1 h in avidin-biotin complex solution (ELITE ABC kit, Vector Laboratories, Inc.; 4.5 µl each/ml incubation mixture). After rinsing first in PBS and then in 0.175 M sodium acetate (NaOAc), the Fos antibody-peroxidase complex was visualized with a solution of NiSO4 (25 mg/ml), 3,3-diaminobenzidine HCl (NiDAB; 0.2 mg/ml), H2O2 (0.83 µl of a 3% solution/ml final mixture) in aqueous 0.175 M NaOAc that yielded a blue-black reaction product. After approximately 15–20 min, the tissue was transferred into the acetate solution to stop the reaction, rinsed in PBS, and then placed into anti-LHRH (LR-1, gift from Drs. Benoit and Guillemin, 1:100,000 for fluorescence double labeling using amplified biotin techniques or 1:150,000 for conventional double immunoperoxidase labeling) using the protocols described by Lee et al. (31) and Hoffman et al. (20), respectively. The former provided a means by which the pePOA and LHRH activation patterns could be independently determined; the latter offered a means of retaining permanently stained sections. After completion of staining, sections were rinsed in normal saline, mounted onto subbed slides, dried overnight, dehydrated through alcohols, cleared, and coverslipped.

Determination of the FAu injection site. Staining for FAu was accomplished using single immunoperoxidase methods on a 1 in 12 series of sections. The strategy was essentially the same as that outlined above for Fos, but with anti-FAu antibody rather than anti-Fos. Anti- FAu, a gift from Dr. H. T. Chang, University of Tennessee (Memphis, TN) (32) and later purchased from Chemicon (Temecula, CA), was used at a concentration of 1:50,000, in PBS with 0.4% Triton X-100, for 48 h 4 C. After staining for approximately 15–20 min in NiDAB solution, the tissue was transferred into the acetate solution to stop the reaction, rinsed in normal saline, and mounted onto subbed glass slides. The sections were dehydrated through alcohols, cleared in Histoclear, and coverslipped with Histomount. Some sections were counterstained with neutral red to aid in cytoarchitectonic nuclear identification or were double labeled with LHRH (using the same strategy as the Fos/LHRH studies) to better determine whether the LHRH population was encompassed by the injection.

Fos and retrograde tracing. To determine whether retrogradely labeled pePOA neurons were activated at the time of a LH surge, sections (from a 1 in 6 series of sections) were double stained for Fos and FAu. In this instance, Fos was stained first using the immunoperoxidase procedure described previously for Fos and LHRH, with NiDAB as the chromogen. Next, FAu was stained with immunoperoxidase procedures and diaminobenzidene (DAB) as the chromogen (as described above for LHRH) or with biotin-amplified immunofluorescence (33) using a streptavidin-bodipy fluorophore. The latter method consisted of the following steps: after reacting the sections for Fos using NiDAB, the tissue was rinsed in acetate solution followed by a few rinses in PBS and incubated in anti-FAu antibody (1:70,000) for 48 h at 4 C. The tissue was rinsed in PBS and incubated in biotinylated goat antirabbit serum (Vector Laboratories, Inc.; 1:5,000 in PBS with 0.4% Triton X-100) for 1 h at room temperature. After rinsing, the tissue was incubated for 30 min with ELITE avidin-biotin complex reagents (1.125 µl each/ml mixture in PBS with 0.4% Triton X-100), rinsed again for 30 min, and incubated for 15 min in biotinylated tyramine [prepared as previously described (34)] to which H2O2 was added to achieve a final concentration of 0.005%. The tissue was rinsed for 30 min in PBS and then incubated in streptavidin-Bodipy (5 µl streptavidin-Bodipy/ml PBS with 0.4% Triton X-100) for 2.5 h at 37–40 C. The tissue was rinsed in saline, mounted, air-dried overnight, cleared in xylenes, and coverslipped in Histomount (Fisher Scientific, Rockford, IL).

Triple labeling of Fos, LHRH, and FAu. In a few cases we performed triple labeling of sections to verify that the FAu injections encompassed regions containing activated LHRH neurons, and that the activated pePOA neurons projected to the vicinity of LHRH neurons, as determined by the presence of FAu within Fos-positive pePOA neurons. For this procedure (35) the nuclear antigen Fos is first stained using immunoperoxidase methods and NiDAB as the chromogen, as described above. Transmitter (in this case, LHRH) is next stained with an immunofluorescence-alkaline phosphatase procedure described by van der Loos (36). Briefly, after staining for Fos and incubating in anti-LHRH (1:70,000), the tissue is rinsed and then incubated with goat-antirabbit IgG conjugated to alkaline phosphatase (Vector Laboratories, Inc.; 1:500 for 2 h at room temperature), rinsed in Tris buffer (pH 8.0) for 15 min, and incubated with CAS Red solution (Cell Analytical Systems, Elmhurst, IL) containing levamisole (1 mM) according to manufacturer inserts. [It should be noted that Cell Analytical Systems is no longer making CAS Red; Vector Red (Vector Laboratories, Inc.) prepared according to product inserts can be substituted.] After a 1- to 3-h incubation in the staining solution, the tissue is rinsed in distilled water followed by PBS, incubated in anti-FAu (1:70,000), and processed in the fashion described for biotin-amplified immunofluorescence above (using streptavidin-Bodipy as the fluorophore). After the final reaction, the tissue was rinsed in PBS mounted from saline, air-dried overnight, dehydrated through ascending alcohols, cleared in xylenes, and coverslipped with Histomount. The presence of FAu was evident as a green fluorescent product; LHRH immunoreactivity within the cell cytoplasm was barely detected as pink reaction product with brightfield optics, but was brightly red/orange fluorescent under fluorescence optics; Fos was stained blue-black. LHRH neurons were considered within the area of FAu uptake if diffuse FAu product surrounded the LHRH cells.

LHRH and anterograde tracer, neurobiotin. Labeling of neurobiotin was accomplished immunocytochemically with antibiotin antibodies. Briefly, after rinsing the cryoprotectant from the tissue, the sections (from a 1 in 6 series of sections) were treated with sodium borohydride, rinsed, and incubated with antibiotin (1:60,000; Vector Laboratories, Inc.) for 48 h at 4 C. After incubation with the antibiotin, the tissue was rinsed in PBS, and the sections were incubated in biotinylated rabbit antigoat IgG and avidin-biotin complex reagents as described above. Sections were then rinsed in PBS followed by NaOAc solution. The staining of neurobiotin was accomplished by using a mixture of H2O2 (0.83 µl of a 3% solution/ml reaction solution) and NiDAB. After staining for 7–10 min, the tissue was transferred to acetate solution to stop the reaction, rinsed in PBS, incubated in anti-LHRH (1:150,000), and stained with either DAB or amplified fluorescent labeling as described for the Fos and LHRH double labeling above. Axons labeled for neurobiotin appeared blue-black; LHRH was stained either brown when DAB was used or fluoresced red with the fluorophore, Texas Red.

Tissue analysis
Retrogradely labeled cells or cells stained for Fos and/or LHRH and the extent of the tracer injection were plotted using stage-mounted X-Y potentiometers mounted on a Nikon fluorescence microscope (Melville, NY) linked to a Macintosh II computer (37).

Analyses of the interactions of neurobiotin-containing axons and LHRH were qualitative. The criteria for considering a LHRH neuron contacted by a neurobiotin-positive axon were those established in the report by Fitzsimmons et al. (38) and required that no visible space be present interposed between the biotin-immunoreactive axon and the LHRH neuron at a magnification of x600.

LH RIA
The RIA protocol we used for plasma LH has been previously described (39, 40). Plasma volumes of 20 and 5 µl from each animal were assayed for each sample. For the LH assay, the rat LH CSU-120 antibody was used. Standard curves were constructed using the rat LH RP-3 standard from the National Hormone and Pituitary Program of the NIDDK.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fos expression in LHRH neurons and that in pePOA neurons were coordinated during the LH surge
No increased pePOA Fos or LHRH-Fos activation was noted at any time on diestrous I or II or during the morning of proestrus when LH values were low (Table 1Go and Figure 1Go). As expected (27, 28, 29), animals killed on the afternoon of proestrus showed marked expression of Fos within LHRH neurons that first appeared approximately 8 h after lights on when the rising phase of the LH surge was initiated. Each animal displaying an increase in plasma LH values showed activation of LHRH neurons, and there was a coordinated increase in pePOA Fos staining as well. The pePOA Fos pattern at the peak of the LH surge lay within the region 100–150 µm lateral to the third ventricular surface that was maximal 300–450 µm caudal to the OVLT (Figure 2Go). At the rostral pole of the third ventricle (where LHRH neuron numbers are maximal) few Fos-positive cells were found in the periventricular zone (Fig. 2AGo). At the more caudal pePOA levels (Fig. 2DGo), the numbers of Fos-positive neurons declined in the pePOA. By the beginning of the hypothalamus, about 150 µm caudal to level D, Fos-positive cells in the periventricular zone were no longer present (not shown).



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  		Figure 1. Micrographs of Fos expression within pePOA neurons (brightfield images) and LHRH neurons (insets) before (a–c) and during (d) a LH surge. In the rats killed before the LH surge, the LHRH neurons are devoid of Fos, and the pePOA displays few Fos-positive nuclei, whereas at the time of the LH surge (d), LHRH neurons (inset) and numerous neurons of the pePOA (arrows) are activated to express Fos. Bar = 100 µm (brightfield images) and 10 µm (fluorescence images).

 


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  		Figure 2. Rostral to caudal brain plots (A) at the level of the OVLT where LHRH neurons are most numerous and 300 (B), 450 (C) and 600 (D) µm caudal to the OVLT depict LHRH neurons and Fos-positive non-LHRH neurons from an animal killed at the time of the LH surge. Fos-only neurons (small dots) are plotted for a distance of approximately 250 µm from the midline to include the entire pePOA, but not the lateral POA. Open squares indicate LHRH neurons devoid of Fos; filled squares show those LHRH neurons that expressed Fos. Note that the pePOA Fos activation is most marked at levels 300–450 µm from the OVLT.

 
Retrograde tracer injections into regions containing LHRH neurons labeled pePOA neurons, some of which were Fos activated at the time of a LH surge
To determine the likelihood that pePOA neurons activated at the time of a LH surge innervated LHRH neurons, we first made small iontophoretic injections of FAu into the sites of LHRH neurons and then determined whether the retrogradely labeled neurons expressed Fos at the time of a LH surge. Of the 15 animals injected, 7 possessed sites of injection that encompassed LHRH neurons but did not extend into the pePOA. The 7 injection sites were approximately 200–400 µm in diameter. Most were spheres, but in 1 case where the injection lay in the ventrolateral cell field of LHRH neurons, the injection spread caudally with the LHRH population for about 600 µm but did not spread dorsally or medially. In each case where LHRH neurons were present in the injection site, a collection of retrogradely labeled neurons was noted in the pePOA. An example of a typical injection is shown in Fig. 3aGo. Staining of the adjacent section (Fig. 3bGo) for Fos and LHRH revealed that Fos-activated LHRH neurons were present within the injection site and that the pePOA neurons expressed Fos, as expected (Fig. 3cGo). With triple labeling (Fig. 4Go) we could verify that the injection sites possessed activated LHRH neurons.



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  		Figure 3. Micrographs show one of the FAu injections into the region containing LHRH neurons. FAu/LHRH is shown in a; Fos/LHRH in b; more caudally, the pePOA Fos pattern in c; retrograde labeling pattern in d; and double labeling of Fos and fluorogold in e in the same animal. In a, LHRH is brown, and FAu is blue-black. In b, the Fos (blue-black)/LHRH (brown) obtained from the section adjacent to a verifies that the LHRH neurons were activated. The inset shows the side of the injection with two LHRH cells located at approximately the positions indicated by the asterisks in a. In c, note that the pePOA shows Fos activation, and the same region (d) obtained on the adjacent section possesses numerous retrogradely labeled neurons. In e, some of the FAu-labeled neurons (brown cytoplasm) are also Fos positive (blue-black). Bars = 100 µm (a, c, and d) and 10 µm (b and e).

 


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  		Figure 4. Triple labeling of FAu, LHRH, and Fos. FAu is revealed with biotin-amplified fluorescence (green, Bodipy fluorescence); LHRH is stained with a red fluorophore (CAS red); Fos is stained with NiDAB (black). A, The boundaries of the FAu injection are indicated by white arrowheads in a section taken at a level of the OVLT, just rostral to the center of the injection site. The arrow in A indicates one of the LHRH neurons within the injection site. This cell is magnified in B and C. The LHRH cell viewed under fluorescence optics (B) shows the strong immunoreactivity of LHRH. C, Brightfield image of same cell shows the presence of Fos activation (black nucleus) in the LHRH neuron. Bars = 100 µm (A) and 10 µm (B). D, Plot showing the injection site of the same animal depicted in A–C, including plots of the LHRH neurons (circles) and Fos+ neurons that do not express LHRH (small dots). LHRH neurons that are Fos+ are filled circles; LHRH neurons not expressing Fos are open circles. No retrograde labeled cells were seen in that region. E, Retrograde labeling of POA neurons after the injection of FAu into the LHRH cell-containing region is depicted. Squares indicate the FAu cells; open squares lack Fos staining, and closed squares were Fos+. Fos only in the pePOA is shown with small dots. F, Injection site and LHRH activation patterns in another rat (no. 7423). G, Retrograde labeling pattern in the pePOA from rat 7423. The red box shows the region magnified in the inset illustrating one Fos+ retrogradely labeled neuron (white arrow) and above it one of the retrogradely labeled neurons that did not express Fos. Red structures are portions of LHRH neurons (dendrites and axons) in the same region. Bar = 100 µm (A) and 10 µm (B, C, and G).

 
The pattern of retrograde labeling within the pePOA from animals with injections that included LHRH neurons invariably revealed a population of neurons located within the pePOA (Figs. 3dGo and 4Go, E and G). Although in some animals the ventricular surface was infused with FAu, the ependymal lining was generally not torn. The labeling within the pePOA was probably not the result of disruption of the ependymal lining or leakage into the ventricle, as the locations of the retrogradely labeled neurons indicated that the projections from the pePOA to the LHRH neurons were essentially uncrossed and if derived from the ventricle would have been bilateral. In some injections, staining of FAu did not saturate the ependymal cells, indicating that the tracer did not reach the ventricle, and in those animals, the general pePOA FAu labeling pattern was not different from that in animals that did show ependymal FAu saturation.

Measurement of LH from plasma obtained at the time of death and examination of Fos and LHRH staining verified that each of the FAu-injected animals displayed a proestrous LH surge, LHRH Fos activation, and pePOA Fos activation. The presence of the tracer did not hinder the animal’s ability to show Fos activation in either LHRH neurons or the pePOA (as determined by symmetry of staining). Double labeling of Fos and FAu (Figs. 3eGo and 4GGo) established that a subset of the retrogradely labeled pePOA neurons was Fos activated. Although as many as 25–50% of the pePOA retrogradely labeled neurons expressed Fos, the numbers of pePOA neurons that were Fos positive after any one injection comprised only a small fraction of the total Fos-activated pePOA population. In considering this feature, one must be aware that the LHRH neurons are so widely scattered that injections encompassing large numbers of LHRH neurons could not be made without the FAu extending into the pePOA (which lies only 300 µm from the largest collection of LHRH neurons). Thus, as is shown in Fig. 4Go, individual injections, although including regions where LHRH neurons were present, encompassed only small numbers of LHRH neurons, and the double labeled pePOA neurons constituted only a small portion of the total Fos-activated pePOA population (Fig. 4Go, E and G). Nonetheless, the presence of even a few activated retrogradely labeled pePOA neurons demonstrates that sites of LHRH neurons receive projections from the pePOA.

Anterograde labeling verified that projections from the pePOA extended to LHRH neurons
Experiments in which neurobiotin was injected into the pePOA determined that pePOA neurons extended axons to the LHRH neurons. Of the 17 animals injected with neurobiotin, 7 had injections that encompassed the pePOA with little spread into the adjacent POA. Tissue from these animals showed neurobiotin-positive axons arising from the pePOA in close contact with LHRH neurons (Figs. 5Go-7 and cover photo). These encompassed LHRH neurons around the OVLT and more caudal sites, but generally did not involve the rostrally located LHRH neurons. A single axon made multiple contacts on the same LHRH neuron (Fig. 5Go). Two injection sites of neurobiotin into the pePOA are illustrated in Figs. 6Go and 7Go to show the range of the injection sizes we obtained. For each of these, examples of biotin-labeled fibers in contact with LHRH neurons are included (insets). There did not appear to be a preferred dorso-ventral position within the pePOA that affected the number of interactions noted, but injections that were centered away from the ventricular surface with only minimal spread to the pePOA had very few (if any) axonal interactions with LHRH neurons.



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  		Figure 5. Examples of a LHRH neuron contacted by neurobiotin axons. Note that the black neurobiotin axon contacts the LHRH neuron at a number of sites along its dendrite. The arrow in the larger photo points to one of these contacts. In the subsequent micrographs (taken at different focal planes), the small arrow marks that site, and the other sites of apparent contact are numbered. Bar = 10 µm.

 


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  		Figure 6. Micrograph of one of the smaller neurobiotin injection sites that was restricted to the pePOA (A). The insets B and C show juxtapositions of neurobiotin-labeled axons with two LHRH neurons. B1 and B2 illustrate multiple contacts seen at different focal planes. Bar = 100 µm (in the low power micrograph A) and 10 µm (in the inset micrographs).

 


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  		Figure 7. A larger injection with spread more extensively along the rostral caudal axis of the pePOA also labeled axons in contact with LHRH neurons. The insets B and C show juxtapositions of neurobiotin-labeled axons with two LHRH neurons. Bar = 100 µm (in the low power micrograph A) and 10 µm (in the inset micrographs).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of our study indicate that there is a highly coordinated Fos activation of the pePOA and LHRH neurons at the time of a LH surge. Our anatomical data indicate that pePOA neurons project to LHRH neurons, and a subpopulation of pePOA neurons is stimulated at the time of the LH surge. Together, these data raise the likelihood that the pePOA is assuming a role in the regulation of LHRH activity at the time of a LH surge. A modulatory role for the pePOA in the regulation of gonadotropic function is consistent with the results of earlier studies aimed at determining where control of the LH surge was mediated. For example, electrolytic lesions of the rostral preoptic area blocked estrous cyclicity as well as steroid-induced LH surges (10, 11, 12). These lesions either severed any possible connection of pePOA neurons with LHRH neurons or included the pePOA per se (Wiegand, S. J., personal communication). Our study now expands on these reports to indicate exactly where within the POA the focus of control lies.

The data we present also clarify the presence of Fos induction previously observed by Insel (17). In that study, the administration of estradiol to ovariectomized rats induced Fos in pePOA neurons at a very precise time after estrogen treatment. Insel discussed this phenomenon as a consequence of the duration of exposure of the neurons to estrogen. Upon close examination, the time after estrogen treatment that evoked the greatest Fos activation was the interval that probably induced a LH surge. Data we and others obtained earlier indicated that the administration of estrogen with progesterone to immature rats (18) or to ovariectomized adult rats (16, 41) evokes a pattern of pePOA activation when the LH surge was induced that is similar to that seen at the time of a spontaneous LH surge. This feature is true whether estrogen is administered in the morning (immature rats) or at later times before the surge (adults), but is absent if estrogen is given with progesterone in a paradigm that blocks, rather than evokes, the LH surge (18). Taken together, these results indicate that the activation of Fos in the pePOA is the result of events that triggered a LH surge, not simply events resulting from the timing of estrogen treatment.

At least a subset of pePOA neurons projected to the vicinity of LHRH neurons, as determined by retrograde labeling. The fact that only a few activated pePOA neurons were retrogradely labeled after injections into regions containing the LHRH neurons could indicate that the pePOA neurons are involved in multiple functions other than just those influencing LHRH neurons. However, as only a few LHRH neurons were exposed to tracer by any one FAu injection, it is probable that the degree to which the pePOA interacts with LHRH neurons is far greater than that revealed by any one FAu injection. Although such a consideration could explain why only a few of the activated pePOA neurons expressed FAu, the data do not explain why not all of the pePOA neurons that were retrogradely labeled expressed Fos at the time of a LH surge. There are a number of possible explanations for this. First, the pePOA is a heterogeneous population of cells; only a subset of the pePOA cells might serve stimulate LHRH release, whereas others might serve to inhibit, rather than stimulate, LHRH release. Secondly, as the LHRH neurons are not concentrated anywhere in the brain, the retrograde tracer always exposed other neurons that did not contain LHRH. Hence, it is possible that the pePOA is participating in functions other than simply regulation of the LH surge.

Anterograde labeling studies from the pePOA confirmed that the projection to LHRH neurons is direct, although neurons other than LHRH neurons also appeared innervated by the pePOA neurons. Whether any or all of these non-LHRH neurons contribute to LH control is unknown, but investigations have implicated non-LHRH POA neurons containing transmitters such as {gamma}-aminobutyric acid (42), neurotensin (5, 43), and atrial natriuretic peptide (44) in the regulation of LH secretion in females. In some of the larger injections, there appeared to be a greater number of LHRH neurons that were contacted by the POA neurons. Yet, injections that were placed more centrally within the POA away from the ventricular surface had few such interactions, suggesting that indeed the pePOA was providing the significant input to LHRH neurons. As it was not possible to label the entire pePOA without the injection extending more laterally, we are not certain of the true magnitude of the projection to LHRH neurons, and it is unlikely that tract-tracing studies will definitively resolve the issue of the magnitude of control of LHRH neurons by the pePOA. We also acknowledge that the use of anterograde tracing does not prove that the same neurons providing the labeled fibers are the pePOA neurons that expressed Fos at the time of the surge, but it raises confidence that these neurons could provide such input. In a preliminary study we observed that chemical lesion of the pePOA neurons prevents Fos activation of LHRH neurons at the time of a LH surge, further suggesting that the anatomical link described has functional significance.

Although synchronization of pePOA activation with LHRH activation and anatomical connectivity suggest that the pePOA has a role in modulating LHRH activity, what that role must still be defined. Studies from our laboratory indicate that the pePOA neurons activated at the time of a LH surge contain both progesterone receptors (18) and estrogen ({alpha}) receptors (unpublished data). The fact that LH surges can be induced by local exposure of the POA to these steroid hormones suggests that the pePOA is the transduction site for steroidal effects on LHRH activity.

What now remains an important next step is characterization of the phenotype of the pePOA neurons. Preliminary studies (41, 45) have suggested that at least some of the activated pePOA neurons contain dopamine (~10%), whereas others express galanin (~ 40%) and both of these transmitters are implicated in LHRH control (46, 47). Determining the transmitter of the remaining pePOA neurons requires further study.


   Acknowledgments
 
The authors thank Drs. Anne Murphy and Lique Coolen for their careful reading of the manuscript and their constructive editorial comments.


   Footnotes
 
1 This work was supported by Grant NS-28730. Back

Received May 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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