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Endocrinology, doi:10.1210/en.2004-1538
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Endocrinology Vol. 146, No. 4 1940-1947
Copyright © 2005 by The Endocrine Society

Temporal Changes in Gene Expression in the Arcuate Nucleus Precede Seasonal Responses in Adiposity and Reproduction

Alexander W. Ross, Lynn M. Bell, Pauline A. Littlewood, Julian G. Mercer, Perry Barrett and Peter J. Morgan

Molecular Endocrinology Group, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Professor Peter J. Morgan, Institute Director, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom. E-mail: p.morgan{at}rowett.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In anticipation of seasonal climate changes, Siberian hamsters display a strategy for survival that entails profound physiological adaptations driven by photoperiod. These include weight loss, reproductive quiescence, and pelage growth with shortening photoperiod and vice versa with lengthening photoperiod. This study reports gene expression changes in the hypothalamus of Siberian hamsters switched from short days (SD) to long days (LD), and also in photorefractory hamsters. Siberian hamsters were maintained in either LD or SD for 14 wk, conditions that generate physiological states of obesity under LD and leanness under SD. After 14 wk, SD lighting was switched to LD and gene expression investigated after 0, 2, 4, and 6 wk by in situ hybridization. Genes encoding nuclear receptors (RXR/RAR), retinoid binding proteins (CRBP1 and CRABP2), and histamine H3 receptor were photoperiodically regulated with significantly lower expression in SD, whereas VGF mRNA expression was significantly higher in SD, in the dorsomedial posterior arcuate nucleus. After a SD-to-LD switch, gene expression changes of CRABP2, RAR, H3R, and VGF occurred relatively rapidly toward LD control levels, ahead of body weight recovery and testicular recrudescence, whereas CRBP1 responded less robustly and rxr{gamma} did not respond at the mRNA level. In this brain nucleus in photorefractory animals, the CRABP2, RAR, H3R, and VGF mRNA returned toward LD levels, whereas CRBP1 and rxr{gamma} remained at the reduced SD level. Thus, genes described here are related to photoperiodic programming of the neuroendocrine hypothalamus through expression responses within a subdivision of the arcuate nucleus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEASONAL MAMMALS USE a range of strategies involving adaptations in their physiology and behavior to enable them to survive predictable harsh winter conditions (1, 2, 3, 4). Although many accumulate fat stores, the Siberian hamster reduces food intake and sheds body weight (5, 6). These adaptations can be induced under artificial laboratory lighting conditions (7, 8). After transfer from a long photoperiod into a short photoperiod, the Siberian hamster reduces food intake in the face of ad libitum food availability, and this is accompanied by a profound loss of body mass through catabolism of fat stores (6, 9). In addition the animals become reproductively quiescent under short photoperiod (10). An interesting feature of this animal is that it adjusts its body mass according to the duration of exposure to short photoperiod (7, 8).

These physiological changes are reversible, given that low-body-weight hamsters held on short photoperiod regain body weight when transferred back into long photoperiod (5). Furthermore, if animals are maintained on short days (SD) indefinitely, eventually, after about 20 wk, the animals become refractory to the SD signal and spontaneously regain body weight (11, 12). This phenomenon is called photorefractoriness (11, 12, 13, 14, 15).

The photoperiodic signal is transduced into durational nocturnal melatonin secretion, which conveys the seasonal time cue to the neuroendocrine system (16, 17). Melatonin receptor mapping, melatonin infusion studies, pinealectomy, and lesioning studies have shown the importance of the hypothalamus to photoperiodically regulated changes in reproduction and energy balance (17, 18, 19, 20, 21, 22, 23, 24). By contrast, the seasonal regulation of prolactin secretion in sheep involves the pituitary pars tuberalis and appears to be independent of the hypothalamus (25, 26). The regulation of seasonal prolactin in the hamster may also involve the pars tuberalis (27), although there still appears to be a role for the hypothalamus in the Siberian hamster (28).

Since the discovery of neuropeptide Y (NPY) in 1984, followed by leptin a decade later, studies in mice and rats have defined some of the hypothalamic circuits involved in the regulation of food intake and energy expenditure (29). A hypothalamic regulatory network comprising a primary neural axis linking the arcuate nucleus (ARC) to the paraventricular nucleus, with additional communication via the neighboring lateral hypothalamus and ventromedial hypothalamus, together integrate responses to and from peripheral visceral organs involved in energy homeostasis (29). Leptin signaling is transduced via receptors on ARC neurones that coexpress NPY and Agouti-related peptide (AGRP) and neurones coexpressing cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC). High leptin concentrations suppress NPY/AGRP expression and activate POMC/CART expression, generating opposing orexigenic and anorexigenic pathways, respectively (30). Siberian hamsters respond in a manner similar to that of nonseasonal rodents in conditions of energy imbalance. However, when these animals lose weight in response to a photoperiod switch from long days (LD) to SD, the reduced circulating leptin signal does not impact upon these anorexigenic and orexigenic pathways as might be predicted. In SD, NPY and AGRP mRNA would be predicted to increase with reduced leptin, but they do not change (4). ObRb and CART mRNA display opposite responses with ObRb decreasing, whereas CART mRNA increases, and only POMC mRNA changes as expected (4). Thus, Siberian hamsters appear to remain in energy balance despite the SD photoperiod-induced appetite reduction and weight loss, yet they retain the ability to respond to periods of imposed food restriction. However, when the ARC is chemically ablated, while sparing the discrete area of this nucleus described as the dorsal medial posterior ARC (dmpARC), the body weight response to photoperiod change is retained (31, 32). This implies that brain circuits involved in the control of these long-term effects of photoperiod on body weight are distinct from those that modulate acute energy imbalances. The underlying neural control of this seasonal anticipatory mechanism is unknown. Similarly, the timing mechanism of the seasonal gonadal cycle in not understood.

In a previous study, we used microarrays to search for photoperiod-responsive genes in the Siberian hamster hypothalamus that could be involved in driving seasonal adaptations (33). We identified CRBP1 as a potential candidate and subsequently investigated it and related retinoid signaling genes and showed that CRBP1, CRABPII, RAR, and rxr{gamma} were all expressed under photoperiod control in a discrete nucleus of the hypothalamus described as the dorsal tuberomammillary nucleus (33). This nucleus has now been reidentified as the dmpARC of the hypothalamus (32); and in the present study, the location of such a nucleus is also investigated in the mouse and rat. In a recent study (32), we showed that the histamine H3 receptor (H3R) and VGF genes were differentially regulated in this same hypothalamic nucleus. The retinoid receptors, H3R and VGF, have been implicated in the regulation of either body weight or metabolic rate in rodents (34, 35, 36). In the Siberian hamster, 14 wk after transfer into SD, all of these genes showed altered mRNA expression responses when compared with LD levels (32, 33).

In the present study, the temporal changes in mRNA expression of these genes in the Siberian hamster have been assessed in an attempt to further elucidate their roles in mediating seasonal physiological responses such as seasonal body weight changes and gonadal activity. To this end, we have investigated mRNA expression levels after transfer of Siberian hamsters back into LD, after 14 wk in SD. This allowed better temporal discrimination of the changes in mRNA levels relative to the overt changes in physiology in response to altered photoperiod. We postulate that those genes potentially involved in driving either weight regain or testicular recrudescence must increase ahead of, or in parallel with, the physiological response.

Here we show that the genes studied can be grouped into two categories: 1) those that show large amplitude mRNA expression changes that occur relatively quickly, preceding weight regain and testicular recrudescence (thus, the activity of these genes may be driving these physiological adaptations); and 2) those that display lower amplitude changes that occur only after development of the physiological responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental procedures
All procedures were licensed under the Animals (Scientific Procedures) Act, 1986, and had local ethical approval. Male Siberian hamsters (Phodopus sungorus) were drawn from the Rowett breeding colony and were gestated, suckled, and reared in LD photoperiod. Food (Labsure pelleted diet, gross energy, 15.21 MJ/kg; Special Diet Services, Witham, Essex, UK) and water were available ad libitum for all animals. Hamsters used were 4–6 months old and were individually housed at least 2 wk before photoperiod manipulation. All experimental hamsters were weighed weekly. LD photoperiod hamsters were housed in a 16-h light, 8-h dark photoperiod cycle. SD photoperiod hamsters were housed in an 8-h light, 16-h dark photoperiod cycle. In the SD-to-LD photoperiod-switch experiment (from henceforth referred to as switchback), LD-housed hamsters were either maintained in LD or transferred to SD for 14 wk. A group of LD and SD hamsters (n = 5) were killed by cervical dislocation at this time point (wk 0). After 14 wk in SD, the lighting was changed back to LD but with temperature (22 C) unaltered. Groups of animals (n = 5) that had been housed under these altered photoperiod conditions, as well as control LD-housed animals, were then killed after a further 2, 4, and 6 wk. Two photorefractory experiments were performed using essentially identical conditions for each. Siberian hamsters were either maintained for 25 wk in LD as controls (n = 14), or were transferred to SD (n = 14) and maintained for 25 wk before killing. In the first experiment CRABP2, RAR, rxr{gamma}, and H3R gene expression was analyzed. The second experiment examined CRBP1 and VGF gene expression and included LD controls (n = 6) and SD animals (n = 7). Both experiments produced very similar body weight and testicular responses. All experimental hamsters were culled 3 h after lights on (ZT3). To help delineate the dmpARC, its location was investigated in mice and rats. Mice were Aston adult males provided with CRM irradiated stock diet (Special Diet Services) and water ad libitum. Rats were out-bred male Sprague Dawley, provided with Purina chow 5001 (PMI Nutrition International, Nottingham, UK) and water ad libitum. Rats and mice were housed under a 12-h light, 12-h dark photoperiod cycle. All brains were immediately dissected, frozen on dry ice, and then stored at –80 C until required.

Riboprobe templates
Riboprobe templates for the CRBP1, CRABP2, RAR, and rxr{gamma} genes were prepared as described previously (33). To prepare the riboprobe template for VGF, Siberian hamster first-strand cDNA was prepared as described previously (32), and then PCR was performed using the forward primer 5'-KGA AAC CCG CAC GCA CAC GCT GAC and reverse primer 5'-MTC CTC CTC CCC GCC CTC CTC TGT designed against nucleotides 801–824 bp and 1549–1572 bp, respectively, of the rat VGF gene with 5-prime degeneracy for human sequence alignment (GenBank accession number M60525 for rat and NM_003378 for human). The PCR amplification temperatures were 94 C for 45 sec, 60 C for 45 sec, and 72 C for 2 min plus 20 sec, for 30 cycles with a final extension at 72 C for 10 min using Pfu Turbo polymerase (Stratagene, Amsterdam, The Netherlands). The amplification product was ligated into PCR-Script (Stratagene) and then cloned before sequence verification. The H3R cDNA probe was prepared as described previously (32). For in situ hybridization using tissues from the animals collected as described above, templates were linearized with appropriate multiple cloning site restriction enzymes, and the riboprobes were synthesized using 35S-UTP (Perkin-Elmer LAS (UK) Ltd., Beakonsfield, Buckinghamshire, UK) with T3 or T7 polymerases (Promega UK, Southampton, UK) as appropriate. The probe labeling reactions and in situ hybridization protocol have been described previously (33). Coronal sections, including the dmpARC, corresponded to –2.30 to –2.54 mm relative to Bregma (37). The slides were apposed with Kodak BioMax MR film (Sigma-Aldrich Company Ltd., Poole, Dorset, UK) and, where appropriate, were coated with LM-1 film emulsion (Amersham Biosciences UK Limited, Chalfont St. Giles, Buckinghamshire, UK). The distributions and levels of hypothalamic mRNAs were analyzed and quantified by computerized densitometry (Image Pro-Plus software, version 5.5.1; Media Cybernetics, Wokingham, Berkshire, UK) of in situ hybridization autoradiograms, following methods described in detail elsewhere (33, 38, 39).

Statistical analysis
Data from the switchback experiment were analyzed by two-way ANOVA with Tukey test where appropriate. The photorefractory animal data were analyzed by t test. Statistical analysis was performed using SigmaStat statistical software (SPSS Scientific Software, Erkrath, Germany). Results are presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects on gene expression of transferring Siberian hamsters from SD to LD
CRBP1, CRABP2, RAR, rxr{gamma}, H3R, and VGF mRNA responses were investigated in the dmpARC of animals that initially had been housed in SD photoperiod for 14 wk then switched to LD. This time point after 14 wk in SD was referred to as wk 0. Gene expression levels were investigated in the brains of animals killed at 0, 2, 4, and 6 wk after the switch from SD to LD photoperiod. Gene expression levels were compared with animals that had been held on LD for the same relative times.

The body weights of animals transferred from SD to LD did not change significantly up to 4 wk after switchback, but had increased significantly (P < 0.001) by 6 wk, reaching levels similar to those of LD animals (Fig. 1Go). Paired testes weights of the switchback animals were significantly lower than LD control weights (P < 0.001) until wk 6, when these were not significantly different from the LD controls (LD, 580.4 mg ± 95.5; SD, 432.4 mg ± 55.8).



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  		FIG. 1. Photoperiod regulates gene expression in the Siberian hamster dmpARC. A–F, In situ hybridization of CRABP2, H3R, VGF RAR, CRBP1, and rxr{gamma} probes, respectively. Representative areas of hypothalamic coronal sections for each gene are shown, including a single representative LD control (LD) at wk 0, as well as from each time point after photoperiod switch from SD back to LD (LDsw). The dmpARC (arrows) and ependymal layer (arrowheads, E) are indicated. The scale bar in LD control in A represents 1 mm. Graphs show mRNA expression levels with time in switchback animals (closed circles, left axis) and LD controls (open circles, left axis). Results are integrated optical densities expressed as mean percentages of LD control values at wk 0 ± SEM. Additional plots are repeated showing the corresponding mean percentage increase in body weight ± SEM with time relative to wk 0, after photoperiod switch from SD to LD (closed squares, right axis) and paired testes weights ± SEM (open triangles, right off-set axis). Each point represents data from four to five animals.

 
In the dmpARC, there were marked temporal differences in mRNA expression profiles for each gene studied, in response to the switch from SD to LD. CRABP2 mRNA levels in SD were significantly lower than LD levels at the time of switch (P < 0.05, Fig. 1AGo). The levels of CRABP2 mRNA in the dmpARC increased relatively rapidly after switchback; because, by 2 wk, the levels were not only significantly higher than the levels at wk 0 (P < 0.001) but they were also similar to LD levels, reaching a plateau maintained at the 4 and 6 wk time-points. This was approximately 3-fold higher than LD levels (Fig. 1AGo, wk 4 and 6, P < 0.001).

The response of H3R mRNA expression was similar to that of CRABP2. At wk 0, H3R mRNA levels were significantly lower than LD controls (P < 0.001, Fig. 1BGo). These mRNA levels increased steadily after switchback and reached levels not significantly different from LD levels after 2 wk. The H3R mRNA expression continued to rise and was significantly higher than LD levels, by approximately 50%, after 6 wk (P < 0.01, Fig. 1BGo).

In contrast to CRABP2 and H3R, VGF mRNA levels in the dmpARC were significantly higher than LD control levels at the time of switch (wk 0; P < 0.001, Fig. 1CGo). After switchback, the levels of VGF mRNA decreased rapidly, to become significantly lower than wk 0 levels by 2 wk (P < 0.01). By wk 4, VGF mRNA levels had returned to levels similar to the LD controls, and these were sustained thereafter (Fig 1CGo). Although the direction of change in gene expression for CRABP2, H3R, and VGF varied, in each case the temporal changes in expression occurred in advance of any detected change in either body weight or testes sizes.

For RAR, the mRNA levels in the dmpARC were significantly lower in SD animals at wk 0 (time of switchback) than in LD controls (P < 0.001, Fig. 1DGo). After switchback, these levels increased steadily, to become significantly higher than wk 0 levels after 4 wk (P < 0.001), and returned to LD levels by 6 wk.

In contrast to the other genes, both CRBP1 and rxr{gamma} gene expression in the dmpARC were relatively unperturbed after the SD-to-LD switchback, despite there being a clear effect of photoperiod after 14 wk in SD (P < 0.001, Fig. 1EGo; P < 0.001, Fig. 1FGo). Only 6 wk after switchback were the mRNA levels of CRBP1 significantly increased, although the levels reached were only about 50% of LD levels (P < 0.001, Fig. 1EGo). The CRBP1 mRNA levels also increased similarly in the ependymal layer of the 3rd ventricle in switchback animals (Fig. 1EGo).

The SD-to-LD switchback also had an effect on VGF mRNA expression in the more ventral and rostral region of the ARC distinct from the dmpARC. However, in this case, the response was opposite to that observed in the dmpARC. The levels of VGF mRNA in the ventral/rostral ARC were lower at wk 0–4 (P < 0.05) in switchback animals than in LD controls. However by 6 wk, expression increased so that it was not significantly different from LD controls (Fig. 2Go). The time-course of this change was coincident with both body weight recovery and testicular recrudescence.



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  		FIG. 2. Photoperiod regulates the expression of VGF mRNA in the Siberian hamster ventral/rostral ARC. In situ hybridization results show that VGF mRNA levels are decreased in SD; but after photoperiod switch to LD, mRNA levels return to LD levels after 6 wk. The graph, including paired testes and body weight plots, is presented as described in Fig. 1Go. Each point represents data from four to five animals.

 
In the hypothalamus of LD control animals, there were no significant changes in the expression of any of the genes considered throughout the 6-wk period of the study.

Gene responses in photorefractory animals
To assess the responses of animals that had become refractory to the SD signal, animals that had been maintained in SD for 25 wk were compared with LD controls. Relative to LD control animals, the body weights of animals maintained in short photoperiod for 25 wk decreased, reaching a nadir at around 19 wk (Fig. 3AGo). By 25 wk in SD, most hamsters had regained more than 25% of the total SD body weight loss. However, it is important to note that some animals had not yet become photorefractory at this time-point (Fig. 3BGo). Paired testes weights increased in proportion to body weights. The mean paired testes weight was significantly lower in the photorefractory animals after 25 wk (P < 0.01, data not shown).



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  		FIG. 3. Body weights of Siberian hamsters maintained in LD (closed circles) or SD (open circles) for 25 wk. A, Results are representative from two experiments, showing the typical body weight nadir after 18–20 wk in SD, which is followed by an increase toward LD levels. Data points show mean weekly body weights ± SEM, with 14 animals per photoperiod. B, Results are representative from two experiments, showing the range of body weight regain after 25 wk, presented as a percentage of the total body weight loss in SD (n = 14 SD animals).

 
For comparison, data from the SD 25-wk photorefractory animals were compared with the 14-wk SD switchback (wk 0) animals replotted from Fig. 1Go. In 25-wk photorefractory animals, CRABP2 mRNA expression in the dmpARC had increased and reached a slightly, but significantly, higher level than LD controls (P < 0.05, Fig. 4AGo). This contrasts markedly with the low levels of CRABP2 mRNA levels observed after only 14 wk in SD, where the CRABP2 mRNA expression was substantially lower than LD controls (P < 0.001, Fig. 4AGo).



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  		FIG. 4. Differential gene expression in the dmpARC in photorefractory Siberian hamsters after 25 wk in SD compared with LD controls. A–F, In situ hybridization using CRABP2, H3R, VGF, RAR, CRBP1, and rxr{gamma} probes, respectively. Bar charts represent quantification of mRNA levels after 25 wk and include data from wk 0 switchback animals maintained in LD or SD for 14 wk. Values at each time point are expressed as mean percentages of the LD control levels ± SEM (***, P < 0.001; *, P < 0.05; NS, no significant difference).

 
A similar, although not identical, response to CRABP2 was seen for H3R mRNA levels. H3R mRNA levels in the dmpARC had increased from relatively low levels after 14 wk in SD, compared with LD levels (P < 0.001), to reach levels that were not significantly different from the LD controls after 25 wk (Fig. 4BGo).

In contrast to CRABP2 and H3R, VGF mRNA in the dmpARC decreased after 25 wk in SD, relative to 14 wk in SD (Fig. 4CGo), reaching levels not significantly different from LD controls (P < 0.001, Fig. 4CGo).

For RAR, CRBP1, and rxr{gamma}, the levels of mRNA expression in the dmpARC, relative to LD controls, were similar for both the 25- and 14-wk time-points, with these being lower in SD (Fig. 4Go, D, E, and F, respectively).

VGF mRNA expression in the more ventral and rostral region of the ARC was unaffected by extended exposure to SD, given that the mRNA levels for VGF in this region of the ARC were similar after both 14 or 25 wk in SD, each being lower by a similar magnitude than LD controls (P < 0.01, Fig. 5Go). This represents an important difference in response between the two distinct regions of the ARC (i.e. dmpARC vs. ventral/rostral ARC).



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  		FIG. 5. Photoperiod regulates the expression of VGF mRNA in the photorefractory Siberian hamster ventral/rostral ARC. In situ hybridization results show that VGF mRNA levels are decreased in SD after 14 wk, and the difference between LD and SD levels remains similar after 25 wk in SD (**, P < 0.01).

 
Comparison of RXR{gamma} mRNA expression in the dmpARC of the Siberian hamster hypothalamus with similar hybridization in mouse and rat hypothalami
In situ hybridization was used to compare the locus of rxr{gamma} expression, described as the dmpARC in the Siberian hamster, with the corresponding loci in this area of the mouse and rat hypothalamus (Fig. 6Go). Dark-field film emulsion-coated slides show that rxr{gamma} mRNA is expressed in a similar location in mouse and rat as in the Siberian hamster.



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  		FIG. 6. rxr{gamma} mRNA is expressed in the dmpARC of the Siberian hamster and in corresponding regions in rat and mouse. In situ hybridization shows RXR{gamma}, as silver grains on film emulsion-coated coronal brain sections, over cells of the dmpARC. A–C, Showing light field sections of hamster, rat and mouse respectively. The dashed line (A) encloses the area referred to as the hamster dmpARC and analyzed for gene expression. D–F, Corresponding hamster, rat, and mouse dark-field images, respectively, are shown. Arrows show the dmpARC, and 3V indicates third ventricle. Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
After transfer of Siberian hamsters from SD to LD, the CRABP2, H3R, and VGF genes show changes in expression in the dmpARC of the hypothalamus that occur in advance of overt changes in physiology, whereas CRBP1 and rxr{gamma} respond more slowly. Changes in RAR appear to occur in parallel with the changes in physiology. In addition, after an extended duration in SD (25 wk), when Siberian hamsters are becoming photorefractory, CRABP2, VGF, and H3R mRNA expression changes in the dmpARC resemble those observed in switchback animals. These results strengthen the view that the dmpARC is an important center involved in the integration of photoperiodic information and which mediates seasonal responses in mammals (32, 33).

The genes investigated in this study may be involved in the regulation of both seasonal body weight change and testicular recrudescence. Because the changes in CRABP2, H3R, and VGF mRNA levels temporally preceded, and RAR mRNA increased in parallel with, the changes in body weight and testicular recrudescence in switchback hamsters, potentially these genes may be involved in determining the physiological responses. By contrast, the slow gene expression responses for CRBP1 and rxr{gamma} indicate that these cannot be rate-limiting determinants of the body weight or testicular responses in the switchback or photorefractory hamsters. However, it remains possible that the mRNA for rxr{gamma} and CRBP1 may be above a minimum level of expression so that the regulation of protein, rather than gene mRNA expression, is the critical factor determining the responses.

Among the retinoid signaling-related genes, CRABP2 showed the most dramatic response, changing from being barely detectable in SD to higher than LD levels within 2 wk of switchback and then being sustained at significantly higher levels than LD for the duration of the study. CRABP2 reportedly channels retinoic acid to nuclear retinoic acid receptors, where it also acts as a coactivator of transcription via these receptors (40). Thus, the all-or-nothing CRABP2 mRNA response observed in Siberian hamsters may be a critical photoperiod-induced switch controlling signaling via the retinoid-related receptors. The CRABP2 mRNA levels higher than controls may indicate that expression of this gene is a rate-limiting step in the translocation of retinoic acid to the nucleus. The contrasting decrease in VGF mRNA levels clearly discounts the possibility that the switch of photoperiod to LD is merely reflected by a general increase in overall transcription levels from more quiescent SD-suppressed levels in this brain locus.

It was also notable that in photorefractory hamsters, CRABP2 mRNA increased to a level above that of LD controls and VGF levels had decreased to LD levels before any major change in either body weight or testes sizes. Thus, in photorefractory animals, CRABP2, VGF, and H3R mRNA expression changes may also be implicated in adjusting the physiology to the LD phenotype. In contrast, the RAR, CRBP1, and rxr{gamma} responses in photorefractory animals suggest that these slow-responding genes either remain under regulation by the SD melatonin signal or simply return to LD mRNA levels at a slower rate than the other genes studied. However, it may be that the levels of protein expression rather than mRNA, for RAR, RXR{gamma}, and CRBP1, are critical to the photorefractory response.

The SD-induced changes in gene expression did not occur in pinealectomized Siberian hamsters; therefore, these changes are most likely dependent upon melatonin (32, 33). There is no evidence that the dmpARC expresses melatonin receptors or has direct photic inputs in the hamster (Ellis, Mercer, and Morgan, unpublished observations). It is possible that some melatonin-target sites, including the suprachiasmatic nucleus of the hypothalamus, nucleus reunions, or paraventricular nucleus of the thalamus, communicate to the dmpARC, because melatonin implants placed at each of these sites were capable of inducing testicular regression in Siberian hamsters (14). In photorefractory animals, the CRABP2, VGF, and H3R genes appear to have become dissociated from the normal SD influence on gene transcription; and thus, control in this nucleus is likely to lie downstream of a melatonin target site. In the photorefractory animals, these genes had become refractory to the extended photoperiod by 25 wk, but the physiological responses in these animals were asynchronous.

The retinoid signaling response to altered photoperiod may be gated at several levels; one mechanism may involve activation through a potentially higher concentration of ligand presented to the retinoid receptor by the increased level of CRABP2. In addition, the elevated CRABP2 level could increase CRABP2-mediated coactivation of transcription (41, 42). Furthermore, an as-yet-unidentified binding partner of RXR{gamma} and/or coactivators of RXR{gamma} may be involved in driving downstream responses. Identification of genes targeted by retinoid signaling in the dmpARC is an important step toward revealing downstream responses, and this may be aided by consideration of candidates evaluated in a recent study of 1191 papers, covering 532 genes reportedly regulated by retinoic acid (43).

Both VGF and the histamine H3 receptor were included in the present study because, in an earlier study, we showed that these genes were dynamically expressed in the dmpARC (32). The mRNA expression of H3R was also shown to colocalize on cells that expressed rxr{gamma} in the dmpARC. Results from the previous study also indicated that after 14 wk after transfer of both Siberian and Syrian hamsters from LD to SD, VGF mRNA levels increased, whereas H3R levels decreased. For the H3R, at least, this was shown to be truly photoperiod-induced, because H3R mRNA expression, in animals that were pinealectomized before transfer to SD, displayed LD levels and was thus dependent upon a pineal secretion, most likely to be melatonin (32). Here we have extended these findings by showing that both VGF and H3R are among the fast-responding genes in the dmpARC, having returned to their respective low and high mRNA levels, before the physiological changes in body weight and reproductive competence, after SD-to-LD transfer. Histamine was considered interesting because it may be a central component of a hibernation mechanism, an alternative seasonal strategy used by some mammalian species to minimize energy expenditure over winter (44). Histamine has also been implicated in the regulation of both food intake and reproduction in nonseasonal species (45). VGF is abundantly expressed in the brain. It is also found in the pituitary, adrenal, gut, and pancreatic tissues (34). Importantly it has been implicated in the regulation of both energy balance and reproduction, but the mechanism of action of VGF is unclear. Although VGF is a large 80- to 90-kDa protein, smaller peptide products are produced through proconvertase cleavage, which are then stored in dense core granules and are therefore secreted products. However, it remains to be clarified whether VGF functions solely in the process of secretion or whether its cleavage products give rise to biologically active peptides (34) or both. In the Siberian hamster, changes seen in VGF mRNA levels in the dmpARC contrasted with those in the ventral/rostral ARC. In the dmpARC, the levels changed from high levels in SD to low levels in switchback animals and also to low levels in photorefractory animals. In the ventral/rostral ARC, however, VGF mRNA levels increased from low levels in SD to high levels in switchback animals and did not become refractory to the extended SD. In the ventral/rostral ARC of fasted mice, elevated VGF expression can be inhibited by exogenous leptin injection (46). Siberian hamsters express leptin receptors in the ARC, with decreased levels observed in SD compared with LD animals (4). Siberian hamsters also are more sensitive to exogenous leptin in SD than in LD (4). Thus, in the more sensitive SD environment, leptin may exert suppression on VGF expression in the ventral/rostral ARC. In photorefractory animals, leptin sensitivity may have changed insufficiently toward the less sensitive LD form to allow elevated VGF expression in the ventral/rostral ARC. These differential VGF expression responses in the ventral/rostral ARC, compared with the dmpARC, suggest the possibility of more than one role for VGF or its metabolic products.

In a previous study we described the locus of photoperiod-sensitive gene expression as the dorsal tuberomammillary nucleus (33); but in the light of more recent data, this interpretation has been revised to the dmpARC (32). The site of expression of the retinoid genes in the Siberian hamster corresponds with the position of the dmpARC. In addition, the location of rxr{gamma} mRNA expression in the dmpARC in the Siberian hamster was compared, and found to correspond, with similar sites of rxr{gamma} mRNA expression in mouse and rat. The role of rxr{gamma} in this region in the mouse and rat remains unclear. However, in photoperiod responsive animals, the dmpARC is a region of the brain where an increasing number of photoperiodically regulated gene expression changes have been observed. Further characterization of this region may allow the classification of this region as a new and functionally important subdivision of the ARC in seasonal, as well as nonseasonal, mammals.

The results presented here provide support for the previous investigations of retinoid-related genes as well as VGF and H3R (32, 33) and provide new information on genes related to photorefractoriness, suggesting novel roles for these in mediating seasonal physiological responses. Changes in thyroid binding protein genes have also been implicated in Siberian hamster photorefractory responses, but the precise location of their expression within the hypothalamus has not been defined (47). The close apposition anatomically of the dmpARC to the ARC suggests that it is strategically placed to interact with those regions of the ARC involved in acute responses to energy demand and in the long-term maintenance of seasonal body weight. Further studies are required to elucidate the neural interconnections and communications to and from the dmpARC and to delineate the functional effects of the gene expression changes presented.


   Acknowledgments
 
The authors thank Dr. Z. Archer for providing rat brain tissues, and Tracy Logie for animal husbandry.


   Footnotes
 
This work was supported by the Scottish Executive Environment and Rural Affairs Department.

First Published Online January 6, 2005

Abbreviations: AGRP, Agouti-related peptide; ARC, Arcuate nucleus; CART, cocaine and amphetamine-regulated transcript; dmpARC, dorsal medial posterior ARC; LD, long day(s); NPY, neuropeptide Y; POMC, proopiomelanocortin; SD, short day(s).

Received November 29, 2004.

Accepted for publication December 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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