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Biology of Reproduction 61, 1115-1122 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

A Morning Surge in Plasma Luteinizing Hormone Coincides with Elevated Fos Expression in Gonadotropin-Releasing Hormone-Immunoreactive Neurons in the Diurnal Rodent, Arvicanthis niloticus1

Teresa L. McElhinnya,b, Cheryl L. Siska,c, Kay E. Holekampb, and Laura Smale,a,b,c

a Departments of Psychology and Zoology b and the Neuroscience Program, c Michigan State University, East Lansing, Michigan 48824


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arvicanthis niloticus is a diurnal murid rodent from sub-Saharan Africa. Here we report on processes associated with mating in this species in an attempt to elucidate how the neural mechanisms governing temporal organization differ in nocturnal and diurnal species. First, we systematically mapped the distribution of GnRH neurons in adult females. Second, we tested the hypothesis that Arvicanthis differ from nocturnal murid rodents with respect to the timing of the LH surge and the associated increase in Fos expression in GnRH-immunoreactive (IR) neurons. We examined these events around a postpartum estrus. When parturition occurred between zeitgeber time (ZT) 2 and 17 (lights on at ZT 0 and off at ZT 12; there are 24 ZT units a day, each equivalent to 1 standard hour), we collected blood and perfused females at ZT 17, 20, 23, or 2. A sharp peak in plasma LH occurred at ZT 20, and a 10-fold increase in the percentage of GnRH-IR neurons that expressed Fos-IR occurred between ZT 17 and 20. By contrast, this rise occurs in nocturnal rodents during the last few hours of the light period. This is the first indication of a difference between nocturnal and diurnal animals with respect to neural mechanisms associated with a precisely timed event of known significance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many different reproductive behaviors and their physiological correlates are strongly influenced by time of day. For example, female lab rats (Rattus norvegicus) and hamsters (Mesocricetus auratus) experience an ovulatory surge in LH shortly before dark on the day of proestrus, and mate a few hours later [1]. Several lines of evidence suggest that an endogenous circadian timekeeping mechanism plays an important role in the temporal coordination of both mating and the LH surge in these species [2, 3]. For example, delays of the estrous cycle, whether spontaneous or induced by a variety of manipulations, always last approximately 24 h, or one circadian period [4, 5]. Furthermore, ovulation remains tightly coupled to free-running activity rhythms in Rattus when the animals are released into constant light [6]. Precisely timed daily proestrus-like LH surges also occur in ovariectomized female Rattus and Mesocricetus implanted with estradiol-filled silicone elastomer capsules [7, 8]. Thus, daily surges occur in a relatively constant hormonal milieu and are, therefore, not merely a response to changing patterns of ovarian hormone secretion over the course of the estrous cycle. More likely, in intact female Rattus and Mesocricetus, the LH surge is regulated by an interaction between rising levels of estradiol and an internal circadian-gated signal [9].

The suprachiasmatic nucleus (SCN) functions as a clock that governs a wide range of endogenous circadian rhythms in mammals [10]. Several lines of evidence suggest that a signal emanating from the SCN plays an important role in the timing of the LH surge in Rattus and Mesocricetus. In these animals, SCN lesions block the LH surge [11], and knife cuts that sever rostrally directed SCN efferents can interfere with ovulation [12, 13]. The SCN may influence the LH surge via projections onto hypothalamic GnRH-immunoreactive (IR) neurons [14, 15]. Evidence supporting this assertion comes from studies of Fos, a phosphoprotein involved in gene regulation that can reflect levels of neuronal activity. On the day of proestrus, GnRH-IR neurons exhibit a peak in Fos-IR expression during the LH surge in Rattus [16, 17] and Cavia porcellus (guinea pigs) [18], and immediately after the surge in Mesocricetus [19]. Many of the GnRH-IR neurons that express Fos-IR around the time of the LH surge are directly innervated by the SCN [15]. Taken together, these data suggest that a circadian signal influencing the LH surge may be mediated by a direct monosynaptic pathway from the SCN to GnRH-IR neurons in the preoptic area (POA) [15].

To date, research on the neural mechanisms controlling the timing of the LH surge has focused on nocturnal rodents, and nothing is currently known about how these mechanisms might differ between diurnal and nocturnal species. In fact, at a more general level, the neural mechanisms responsible for behavioral and physiological differences between nocturnal and diurnal species remain a mystery. One reason is that there has not been a suitable diurnal rodent with which to experimentally examine the issue. Arvicanthis niloticus is a predominantly diurnal murid rodent from sub-Saharan Africa that represents an ideal model with which to investigate the neural control of circadian-gated reproductive mechanisms. Mating is a very precisely timed event in these animals, occurring approximately 1–2 h before lights-on [20]. Thus, mating occurs about 10 h out of phase in Arvicanthis as compared to nocturnal murid rodents. In the work reported here, we examined the temporal pattern of neural and endocrine events associated with mating in Arvicanthis in an effort to determine how these mechanisms differ in nocturnal and diurnal species. Specifically, we examined the possibility that the temporal pattern of Fos-IR expression within GnRH-IR neurons might be reversed in Arvicanthis from the pattern observed in nocturnal rodents.

The pattern of estrus in nulliparous Arvicanthis is unclear. Corpora lutea are relatively uncommon in ovaries of adult nulliparous females, and vaginal smears provide no evidence of a consistent cycle [21]. Thus, although some individuals sometimes ovulate spontaneously, our only way of assessing when a spontaneous ovulation has occurred is to examine ovarian histology. However, postpartum estrus, which occurs approximately 1 h before lights-on [20], is a highly predictable event in this species and is usually associated with a successful pregnancy. The postpartum ovulation is most likely provoked by a surge in LH associated with parturition [22], which is in turn stimulated by the coordinated activity of GnRH neurons. However, it is not yet known when this LH surge occurs, or when GnRH neurons are activated in Arvicanthis. To explore these issues we first characterized the distribution of GnRH-IR neurons within the forebrains of adult male and female Arvicanthis and then examined the pattern of change in circulating LH and in Fos-IR expression within GnRH-IR neurons around the time of postpartum ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The original stock from which all study animals were derived were trapped in July and August of 1993 from two locations within the Masai Mara National Reserve in southwestern Kenya [23]. Study animals were maintained in plastic cages (38 x 34 x 16 cm) with chipped-aspen litter, in rooms kept at 23°C, with a 12L:12D cycle. Animals were provided water and Harlan Teklad rodent chow 8640 (Harlan Industries, Indianapolis, IN) ad libitum and were supplemented with carrots, whole oats, and rabbit chow (Harlan Teklad hi-fiber rabbit diet 7015) once weekly.

Experiment 1. Distribution of GnRH-IR Neurons

During the light period, two adult female and three adult male Arvicanthis were injected with 1 ml equithesin (i.p.; 42.5 g chloral hydrate and 9.72 g sodium pentobarbital in 1 L of a mixture of propylene glycol, 443.4 ml; ethyl alcohol, 115 ml; magnesium sulfate, 21.26 g; and filtered water) and transcardially perfused with 200 ml PBS (0.01 M) followed by 200 ml 4% paraformaldehyde, with 1.4% lysine, and 0.2% sodium m-periodate in 0.1 M phosphate buffer. Brains were postfixed for 8–12 h, switched to 20% sucrose, and then sectioned at 40 µm along a coronal (n = 1 female and 3 males) or parasagittal (n = 1 female) plane with a freezing microtome. Coronal sections were cut from the diagonal band/medial septal region to the caudal end of the hypothalamus. Every fourth coronal section and every other parasagittal section were processed for immunohistochemical detection of GnRH-IR by means of a standard ABC procedure (Vectastain; Vector Laboratories, Burlingame, CA) using the HU4H antibody to GnRH with diaminobenzidene (DAB) as the chromogen. The HU4H antibody was raised in the mouse against synthetic GnRH conjugated to bovine thyroglobulin. HU4H reacts with mammalian GnRH and its deamidated form, and to a lesser extent with chicken 1 GnRH. HU4H does not react with GnRH precursor or with degraded forms of the molecule [24]. Sections were first rinsed for 30 min with 0.01 M PBS and then incubated sequentially in 1) 5% normal horse serum (Vector); 2) mouse anti-GnRH antibody (HU4H, 1:5000; kindly donated by Dr. H.F. Urbanski, Oregon Regional Primate Research Center, Beaverton, OR); 3) biotinylated horse anti-mouse secondary antibody (Vector, 1:200); and 4) avidin-biotin complex conjugated to horseradish peroxidase (Vectastain ABC Elite kit, Vector; 1:55). Sections were then incubated for several minutes in 0.5% DAB with 35 µl 30% hydrogen peroxide in 100 ml Tris buffer. Sections were mounted on gelatin-covered slides that were then coverslipped. A primary deletion control was performed using the protocol described above, but without the HU4H antibody added to the second incubation. No staining was visible in tissue incubated without the HU4H antibody.

Brains of all Arvicanthis were systematically examined, and individuals were found to be very similar with respect to the general features of the distribution of GnRH-IR neurons. Therefore, one female brain was selected, and maps of every other coronal section through the region containing GnRH-IR neurons in this animal were drawn, resulting in a map depicting GnRH-IR neurons in every eighth section. First, a projection scope was used to draw the outlines of each section; then slides were examined using x10 and x40 objectives of a Leitz Laborlux S light microscope (Leitz Wetzlar GBH, Wetzlar, Germany). All GnRH-IR neurons identified with this latter scope were drawn onto the outlines.

Experiment 2. Fos Expression in GnRH-IR Neurons

Mating couples were paired and left together until a litter was born. Mating couples were checked for new litters at zeitgeber time (ZT) 17, 20, 23, and 2. (There are 24 ZT units per day, each equivalent to 1 standard hour, and ZT 0 = lights on.) If a litter was born between ZT 2 and ZT 17, the female was perfused either at the time the litter was discovered (ZT 17, n = 5) or 3, 6, or 9 h later (ZT 20, n = 4; ZT 23, n = 3; ZT 2, n = 3). Control animals were perfused the day after parturition, at ZT 17 (n = 3), ZT 20 (n = 2), ZT 23 (n = 2), or ZT 2 (n = 3).

At the time of perfusion, animals were injected with 1 ml equithesin (i.p.) and fitted with a light-proof "hood" to prevent light exposure during the perfusion process. Blood was collected into a heparinized syringe via cardiac puncture and centrifuged, and plasma was stored at -80°C. Animals were perfused as described above, and 40-µm coronal sections were cut from the septum through at least the caudal extent of the paraventricular nucleus. Sections were subsequently stored in cryoprotectant [25] at -20°C.

Every other section from each animal was processed using a protocol in which Fos-IR was first visualized with a nickel-enhanced DAB product, and GnRH-IR was then stained with DAB alone. Sections were first rinsed for 1 h in PBS and then incubated sequentially in 1) 5% normal goat serum (Vector), 2) rabbit anti-Fos antiserum (Santa Cruz Biotechnology, Santa Cruz, CA; 1:10 000), 3) biotinylated goat anti-rabbit antiserum (Vector; 1:200), and 4) ABC (Elite kit, Vectastain, 1:55). All incubations were performed in PBS with 2% Triton X-100. Sections were then incubated in 2.5% nickel sulfate containing 0.02% DAB and 0.001% hydrogen peroxide in acetate buffer. Staining for GnRH-IR was then performed using the same procedure as described for experiment 1. Sections were stored in PBS until being mounted on gelatin-covered slides.

Sections were separated into anterior, central, and posterior regions, with approximately 4 sections in each of the first 2 regions and 12 in the third. The anterior region included the medial septum and diagonal band of Broca; the central region contained the POA; and the posterior region included the supraoptic nucleus and the lateral hypothalamic area. These regions roughly correspond to areas depicted in Figure 1, a and b (anterior), Figure 1c (central), and Figure 1, d–f (posterior). A projection scope was used to draw a standard set of outlines of each coronal section from one representative brain. Photocopies of these drawings were used as templates on which GnRH-IR neurons were drawn. For each animal, GnRH-IR neurons with and without Fos-IR were identified with a light microscope (Leitz; Laborluz S, x40 objective) and drawn onto the templates. For each of the 3 regions, the numbers of GnRH-IR cells with and without Fos-IR were summed, and the percentage of GnRH-IR cells that expressed Fos-IR was calculated. These percentages were analyzed using t-tests, linear regression, and one-way ANOVA on arc sine-transformed values to determine whether they differed as a function of time of day. Differences were considered significant when P < 0.05.



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  		FIG. 1. Maps of the distribution of GnRH-IR neurons (closed circles) in coronal sections through the brain of one adult female A. niloticus. Every 8th section from rostral (a) to caudal (f) regions of the GnRH-IR-containing portion of the brain is depicted

Plasma LH concentrations were measured by a double-antibody RIA, which we validated for use with Arvicanthis plasma. Mouse monoclonal anti-bovine LH (518B7, lot 4, obtained from Dr. Jan Roser, University of California, Davis) was used as primary antibody. This monoclonal antibody specifically recognizes LH in a wide variety of mammals [26]. Iodinated ovine LH LER 1056-C2 (obtained from Dr. L. Reichert, Albany Medical College, NY) was used as trace, and goat anti-mouse gamma globulin (Antibodies, Inc., Davis, CA) was used as secondary antibody. Primary antibody was diluted in assay buffer (0.5% BSA in 0.01 M PBS) containing 1.5% normal mouse serum; trace was diluted in assay buffer; and secondary antibody was diluted in phosphate-buffered 5% polyethylene glycol. Ovine LH was iodinated enzymatically (Enzymo-Bead; Bio-Rad, Rockville Center, NY) and purified immediately before use on a concanavalin-A Sepharose (Pharmacia, Piscataway, NJ) affinity column, using methyl {alpha}-D-mannopyranoside to displace iodinated LH. Standard-curve tubes were prepared from S26 ovine LH reference preparation obtained from NIDDK. Standard tubes and unknowns (50 µl) were brought to 150 µl volume with assay buffer. Primary antibody (50 µl) and trace (50 µl) were added simultaneously to standard-curve and sample tubes. Tubes were incubated at room temperature for 24 h. Secondary antibody (1 ml of 0.25%) was then added, and tubes were incubated at room temperature for an additional 3–4 h. Tubes were centrifuged and decanted, and the precipitate was counted. Displacement curves generated by assaying increasing volumes (50–150 µl) of plasma pools from gonad-intact male and female Arvicanthis were parallel to the standard curve (Fig. 2). In order to improve assay sensitivity, assay conditions were slightly modified by decreasing the concentration of primary antibody (from 1:500 000 to 1:800 000) and by increasing the amount of trace (from 10 000 to 15 000 cpm in 50 µl). All unknowns were run in a single assay under these conditions, in which primary antibody bound 23% of the 125I-LH in the absence of cold LH, with nonspecific binding at approximately 4%. The minimum detectable concentration of the assay was 0.38 ng/tube (90% bound), and the assay coefficient of variation was 12.1%. Plasma LH values were examined using one-way ANOVA to determine whether they were influenced by time of day. Differences were considered significant when P < 0.05.



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  		FIG. 2. Displacement curves for pooled plasma samples (50–150 µl) from gonad-intact female (closed circles) and male (closed squares) A. niloticus and for the LH reference preparation (solid line). Curves based on plasma pools are parallel to that of the standard


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Large fusiform bipolar GnRH-IR neurons were clearly distinguishable from surrounding tissue (Fig. 3a). Reaction product filled the cell body as well as axons and dendrites, which did not branch extensively. As in other mammals, GnRH-IR neurons were not clustered into distinct nuclei, but were diffusely distributed in a range of forebrain areas (Fig. 3, b and c). No sex difference was apparent in the number or distribution of these neurons, though the sample was too small to rule out this possibility.



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  		FIG. 3. Photomicrographs of GnRH-IR cells and fibers. High power (a) and low power (b, c). ox, Optic chiasm; ot, optic tract. Bars = 100 µm

The regional distribution of GnRH-IR cell bodies was very similar to that previously described in Rattus [27, 28]. In rostral sections, GnRH-IR cell bodies were present in the ventral and horizontal limbs of the diagonal band of Broca and the medial septal nucleus (Fig. 1, a and b). The density of GnRH-IR neurons was highest in sections through the region somewhat rostral to the middle of their distribution (Fig. 1c). Here, GnRH-IR neurons were found in the medial and lateral preoptic nuclei, as well as in the median preoptic nucleus and the medial septal nucleus. In the caudal half of their distribution, GnRH-IR neurons were almost entirely restricted to the lateral POA and the areas dorsal and medial to the supraoptic nucleus (Fig. 1, d–f).

GnRH-IR fibers and terminals were also distributed in a manner quite similar to that previously described for Rattus. These fibers were most heavily concentrated in an area adjacent to the third ventricle (Fig. 3b), where they were oriented caudally. Labeled fibers were also seen extending in a dorso-ventral direction parallel to the third ventricle, and in a lateral direction immediately above the optic chiasm.

Experiment 2

On the day of the postpartum estrus, there was a sharp peak in circulating LH at ZT 20 (one-way ANOVA, F = 8.738, df = 3, P = 0.002; Fig. 4). Specifically, circulating LH jumped from 2.4 ± 3.6 (ng/ml) at ZT 17 to 19.3 ± 3.3 (ng/ml) at ZT 20, and dropped again to 2.1 ± 4.6 (ng/ml) at ZT 23. All of the animals sampled 3 h later, at ZT 2, and on the day after parturition had LH levels below the limit of detectability for the assay.



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  		FIG. 4. Plasma LH (mean ± SEM) in adult females sampled at different phases of the light:dark cycle (indicated at top of graph) after parturition

Single- and double-labeled GnRH-IR cells were easily discernible using the nickel-enhanced immunohistochemical protocol (Fig. 3 vs. Fig. 5). The number of GnRH-IR cells did not vary as a function of time in any of the three brain regions examined (anterior: F = 0.796, df = 3, P = 0.521; central: F = 2.089, df = 3, P = 0.160; posterior: F = 0.793, df = 3, P = 0.525; Table 1). However, the percentage of GnRH-IR cells with Fos-IR was substantially lower in animals sampled at ZT 17 than at the three later time points (Table 2). In each of the three brain regions examined, this percentage increased significantly as a function of time of day (linear regression slope > 0; anterior: P < 0.017; middle: P < 0.029; posterior: P < 0.036; Table 2). For example, in the central region, the proportion of GnRH-IR cells with Fos-IR increased more than 10-fold, from 3.4 ± 2.4% to 49.2 ± 20.2% between ZT 17 and ZT 20. In all three regions the percentage of GnRH-IR cells with Fos-IR was very low in all five animals perfused at ZT 17. This percentage was high (> 50%) in all animals sampled at ZT 23; but at both ZT 20 and ZT 2, one individual had little Fos-IR within GnRH-IR neurons. The overall percentage of GnRH-IR neurons with Fos-IR was significantly lower in animals killed at ZT 17 (n = 5) than in animals killed at the three later time points (n = 10; t = -3.451, df = 13, P < 0.005). The percentage of GnRH-IR cells with Fos-IR did not vary between brain areas (F = 0.6618, df = 2, P = 0.5213). Animals killed the day after parturition showed little Fos-IR expression within GnRH-IR neurons. Specifically, only 2 of the 10 animals had any Fos-IR in GnRH-IR cells, and these 2 animals had Fos-IR in fewer than 6% of GnRH-IR neurons.



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  		FIG. 5. Photomicrographs of a GnRH-IR neuron containing Fos-IR. Bar = 100 µm


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  		TABLE 1. Average GnRH-IR cell number.*


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  		TABLE 2. Percentage of GnRH-IR cells containing Fos.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Forebrain GnRH-IR neurons in Arvicanthis resembled those observed in other mammals in that they were large bipolar neurons (Fig. 3) distributed in a loose network that crossed classic cytoarchitectural boundaries (Fig. 1). At the rostral end of their distribution, diffusely scattered GnRH-IR cells were found in the septum and the diagonal band of Broca. Caudal to this, GnRH-IR neurons became considerably more abundant and relatively concentrated within the medial and lateral POA. The highest concentration of GnRH-IR neurons was found in this region. In the caudal portion of their range, GnRH-IR neurons were gone from the mediobasal hypothalamus and were found instead above the lateral edge of the optic chiasm. This basic distribution is fairly similar to that described in a number of other rodents [27, 29].

Species differ with respect to where along the rostro-caudal axis the highest density of GnRH-IR neurons is found and also in relation to how far these neurons extend in the caudal direction [30]. The functional significance, if any, of this interspecific variation is unclear. In Arvicanthis, GnRH-IR neurons were most dense in the region of the POA, and they were not found caudal to the supraoptic nucleus. Similarly, very few, or no, GnRH-IR neurons are found caudal to this region in some other myomorph rodents (Rattus [28]; Mesocricetus [29]; Phodopus sungoris [dwarf hamster] [31]). By contrast, GnRH-IR neurons extend as far caudally as the arcuate/infundibular nucleus in Mus musculus (mice) [32] and Cavia [27] and in representatives of non-rodent eutherian orders (primates [33]; chiropterans [34]; carnivores [35–37]; artiodactyls and lagomorpha, reviewed in [30]). Interestingly, the circadian system has been implicated in the regulation of the LH surge in the former groups (Rattus [4]; Mesocricetus [3]) but not the latter (e.g., Cavia, Mus).

In postpartum Arvicanthis, a dramatic elevation in circulating LH was detected at ZT 20, 4 h before lights-on (Fig. 4). All individuals sampled at this time exhibited high levels of circulating LH compared to the levels found in females sampled either 3 h earlier or 3 h later. It is not clear whether a given individual was sampled during the ascending or descending phase of the surge. More frequent sampling will be needed to more precisely characterize the surge and interindividual variability with respect to when it occurs. However, these data clearly reveal that the LH surge occurs in the latter half of the dark period, and that LH has returned to baseline levels by 1 h before lights-on, when Arvicanthis typically engage in postpartum mating (Fig. 6; [20]).



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  		FIG. 6. Contrast between A. niloticus and the R. norvegicus with respect to the timing of the LH surge (arrow), mating (hatched horizontal bar), and changes in Fos-IR expression within GnRH-IR neurons (solid lines). Light:dark cycle is indicated by open and solid bars below the graphs. (Data from A. niloticus represent the average percentage of GnRH-IR neurons from all 3 brain regions [this study]. Data on Fos-IR expression in laboratory rats come from van der Beek et al. [15].)

The timing of the postpartum surge in LH in Arvicanthis differed dramatically from previous findings in the nocturnal species R. norvegicus. Specifically, the LH surge associated with a postpartum estrus occurs late in the light period in Rattus, between ZT 7 and ZT 14 (in 12L:12D; [22, 38]), and late in the dark period in Arvicanthis (Fig. 4). In Rattus and Mesocricetus, the circadian system has been implicated in the temporal coordination of parturition [39–41], the postpartum LH surge [22], and postpartum estrus [41]. For example, in Rattus the postpartum LH surge can be delayed by exactly 24 h by a single appropriately timed injection of sodium pentobarbital [22]. This was the first evidence that a circadian mechanism gates the LH surge during the nonpregnant estrous cycle of Rattus [4]. Fox and Smith [22] have proposed that the circadian system interacts with an interval timer in the regulation of the postpartum LH surge, as it does during a nonpregnant estrous cycle. They suggest that neural signals originating from the cervical stimulation associated with parturition interact with signals emanating from the SCN to regulate the timing of the postpartum LH surge [22]. In Arvicanthis the postpartum LH surge appears to be a fairly precisely timed event, but we do not yet know whether the endogenous circadian timekeeping system plays a role in its regulation.

Fos-IR expression in GnRH-IR neurons was low in all Arvicanthis sampled at the earliest time point, ZT 17, but was dramatically elevated in animals sampled at later time points (Table 2). Furthermore, all 10 animals sampled on the day after a postpartum estrus had very low levels of Fos-IR expression in GnRH-IR neurons. Together, these patterns clearly show that increased Fos-IR expression occurred in GnRH-IR neurons around the time of a postpartum surge in LH, in the latter half of the night. Fos-IR expression remained elevated in these neurons at ZT 23 and ZT 2, after circulating LH had returned to baseline levels.

Diurnal Arvicanthis are strikingly different from nocturnal rodents with respect to the time of the LH surge and the periovulatory rise in Fos-IR expression in GnRH-IR neurons (Fig. 6). In nocturnal murids, Fos-IR expression also rises in association with the ovulatory surge, but this rise occurs in the hours before lights-off rather than the hours before lights-on [17, 19, 42–44]. While rats and Arvicanthis are very different with respect to the timing of ovulatory events relative to the light-dark cycle, these two species are very similar with respect to the coordination of these events relative to each other and to the active period of the day. That is, in both species, the LH surge occurs 3–4 h prior to the beginning of the active period and mating, and Fos expression within GnRH neurons rises around the time that LH rises (Fig. 6). This coordination is presumably necessary to ensure that ovulation occurs at the appropriate time relative to mating.

The current study differs from those on other mammals in that we examined the period around a postpartum estrus, whereas other studies of Fos expression have used animals killed at different times around ovulations that were either spontaneous [17, 42], induced by steroid treatments [18, 44–46], or induced by mating [47]. It is possible that changes in GnRH-IR neurons are not the same around a spontaneous and a postpartum ovulation.

The pattern of Fos-IR expression in GnRH-IR neurons in Arvicanthis differs from the pattern observed in another diurnal mammal, the rhesus monkey [48]. In rhesus, Fos-IR was not elevated in GnRH-IR neurons around the time of LH surges induced by various steroid injection protocols. The time of day at which animals were injected and killed was not reported in that study. Witkin et al. [48] suggest that the neural mechanisms responsible for the production of the LH surge in primates may be different from mechanisms operating in species that exhibit a rise in Fos-IR within GnRH-IR neurons around the time of the LH surge.

The time of Fos-IR expression in GnRH-IR neurons relative to the time of the rising phase of the LH surge appears to be very similar in Arvicanthis and Rattus. In Rattus, GnRH-IR neurons start to express Fos-IR as LH titers begin to rise, and around the time of the surge there is a tight correlation between these two variables [15, 17]. In Mesocricetus, by contrast, the rise in Fos-IR expression does not occur until after the surge in LH [19]. In the current study we found the rise in these two variables to be coincident, but if we were to repeat the study with a finer temporal analysis we might detect a difference. In Arvicanthis, elevated Fos-IR expression appeared to outlast the elevated period of LH secretion by at least 3 h. It is not clear when this Fos-IR decreases after a postpartum LH surge.

To summarize, a comparison of data from this and other studies reveals that the timing of mating, the LH surge, and the associated rise in Fos-IR expression in GnRH-IR neurons are dramatically different in Arvicanthis compared to Rattus (Fig. 6). In Rattus, the SCN plays an important role in the timing of the LH surge, apparently via efferents that terminate on GnRH neurons [15, 43]. If this is also the case in Arvicanthis, then our data suggest that a "switch" located within the SCN, or in the GnRH-IR neurons to which it projects, may determine whether a rodent exhibits a nocturnal or diurnal pattern of function in these neurons. Clearly, this model is contingent on a number of assumptions that have not yet been tested in Arvicanthis. For example, we do not yet know in this species whether the LH surge depends on the circadian system, or whether the SCN projects directly onto GnRH-IR neurons, as it does in Rattus. In any case, the reversal between Rattus and Arvicanthis with respect to the timing of the rise in Fos-IR within GnRH-IR neurons represents the first example of a clear difference between nocturnal and diurnal animals in the neural mechanisms associated with a precisely timed event of known functional significance.


   ACKNOWLEDGMENTS
 
We are grateful to Abel Bult, Sandra Rose, Karen Hubbard, Mary Galloway, and Jane Venier for technical assistance and to Henryk Urbanski for donating the HU4H antiserum. We also thank Dr. J. Roser, Dr. L. Reichert, Dr. A.F. Parlow, and the National Hormone and Pituitary Program at NIDDK for providing materials used for the RIA.


   FOOTNOTES
 
1 This research was supported by NIMH grant RO1 MH53433 (L.S.), NIH RO1 HD26483 (C.L.S.), and a grant from the Searle Scholar Program/Chicago Community Trust (K.E.H.). Back

2 Correspondence. FAX: 517 353 1652; smale{at}pilot.msu.edu Back

Accepted: June 1, 1999.

Received: February 18, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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