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There are compelling reasons to study the role of steroids and sex differences in the circadian timing system. A solid history of research demonstrates the ubiquity of circadian changes that impact virtually all behavioral and biological responses. Furthermore, steroid hormones can modulate every attribute of circadian responses including the period, amplitude and phase. Finally, desynchronization of circadian rhythmicity, and either enhancing or damping amplitude of various circadian responses can produce different effects in the sexes. Studies of the neuroendocrine underpinnings of circadian timing systems and underlying sex differences have paralleled the overall development of the field as a whole. Early experimental studies established the ubiquity of circadian rhythms by cataloging daily and seasonal changes in whole organism responses. The next generation of experiments demonstrated that daily changes are not a result of environmental synchronizing cues, and are internally orchestrated, and that these differ in the sexes. This work was followed by the revelation of molecular circadian rhythms within individual cells. At present, there is a proliferation of work on the consequences of these daily oscillations in health and in disease, and awareness that these may differ in the sexes. In the present discourse we describe the paradigms used to examine circadian oscillation, to characterize how these internal timing signals are synchronized to local environmental conditions, and how hormones of gonadal and/or adrenal origin modulate circadian responses. Evidence pointing to endocrinologically and genetically mediated sex differences in circadian timing systems can be seen at many levels of the neuroendocrine and endocrine systems, from the cell, the gland and organ, and to whole animal behavior, including sleep/wake or rest/activity cycles, responses to external stimuli, and responses to drugs. We review evidence indicating that the analysis of the circadian timing system is amenable to experimental analysis at many levels of the neuraxis, and on several different time scales, rendering it especially useful for the exploration of mechanisms associated with sex differences.

Dopamine has been implicated in circadian timing underlying the food entrainable oscillator (FEO) circuitry and overexpression of the dopamine D2 receptor (D2R) in the striatum has been reported to reduce motivation to obtain food rewards in operant tasks. In the present study, we explored both of these mechanisms by examining food anticipatory activity (FAA) in dopamine D2 receptor-overexpressing (D2R-OE) mice under various durations of food availability. First, we noted that at baseline, there were no differences between D2R-OE mice and their littermates in activity level, food intake, and body weight or in circadian activity. Under conditions of very restricted food availability (4 or 6 hr), both genotypes displayed FAA. In contrast, under 8-hr food availability, control mice showed FAA, but D2R-OE mice did not. Normalization of D2R by administration of doxycycline, a tetracycline analogue, rescued FAA under 8-hr restricted food. We next tested for circadian regulation of FAA. When given ad libitum access to food, neither D2R-OE nor controls were active during the daytime. However, after an interval of food restriction, all mice showed elevated locomotor activity at the time of previous food availability in the day, indicating circadian timing of anticipatory activity. In summary, motivation is reduced in D2R-OE mice but circadian timing behavior is not affected. We conclude that an increase in striatal D2R reduces FAA by modulating motivation and not by acting on a clock mechanism.

Androgens act widely in the body in both central and peripheral sites. Prior studies indicate that in the mouse, suprachiasmatic nucleus (SCN) cells bear androgen receptors (ARs). The SCN of the hypothalamus in mammals is the locus of a brain clock that regulates circadian rhythms in physiology and behavior. Gonadectomy results in reduced AR expression in the SCN and in marked lengthening of the period of free-running activity rhythms. Both responses are restored by systemic administration of androgens, but the site of action remains unknown. Our goal was to determine whether intracranial androgen implants targeted to the SCN are sufficient to restore the characteristic free-running period in gonadectomized male mice. The results indicate that hypothalamic implants of testosterone propionate in or very near the SCN produce both anatomical and behavioral effects, namely increased AR expression in the SCN and restored period of free-running locomotor activity. The effect of the implant on the period of the free-running locomotor rhythm is positively correlated with the amount of AR expression in the SCN. There is no such correlation of period change with amount of AR expression in other brain regions examined, namely the preoptic area, bed nucleus of the stria terminalis and premammillary nucleus. We conclude that the SCN is the site of action of androgen effects on the period of circadian activity rhythmicity.

Successful development of antiobesity agents requires detailed knowledge of neural pathways controlling body weight, eating behavior, and peripheral metabolism. Genetic ablation of FoxO1 in selected hypothalamic neurons decreases food intake, increases energy expenditure, and improves glucose homeostasis, highlighting the role of this gene in insulin and leptin signaling. However, little is known about potential effects of FoxO1 in other neurons. To address this question, we executed a broad-based neuronal ablation of FoxO1 using Synapsin promoter–driven Cre to delete floxed Foxo1 alleles. Lineage-tracing experiments showed that NPY/AgRP and POMC neurons were minimally affected by the knockout. Nonetheless, Syn-Cre-Foxo1 knockouts demonstrated a catabolic energy homeostatic phenotype with a blunted refeeding response, increased sensitivity to leptin and amino acid signaling, and increased locomotor activity, likely attributable to increased melanocortinergic tone. We confirmed these data in mice lacking the three Foxo genes. The effects on locomotor activity could be reversed by direct delivery of constitutively active FoxO1 to the mediobasal hypothalamus, but not to the suprachiasmatic nucleus. The data reveal that the integrative function of FoxO1 extends beyond the arcuate nucleus, suggesting that central nervous system inhibition of FoxO1 function can be leveraged to promote hormone sensitivity and prevent a positive energy balance.

The prominent circadian rhythms of the endocrine system are important for our broader understanding of physiology and behavior, in both health and disease. There is now much evidence that disruptions in the circadian domain, due to either external factors such as shift work or internal factors such as sleep disturbances, can lead to physiological and psychological pathologies. There is interest and urgency in understanding how time is coordinated in the body. A master circadian clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. While this is the only tissue in which autonomous rhythms are self-sustaining, the molecular machinery is present in most peripheral cells. Rather than imposing time on the body then, the SCN entrains local clocks in a tissue-specific manner. These peripheral clocks then participate in controlling rhythmic transcription of genes associated with local tissue function. In this chapter, we examine the interplay between brain and peripheral clocks in the control of endocrine rhythms.

Hamsters will spontaneously 'split' and exhibit two rest-activity cycles each day when housed in constant light (LL). The suprachiasmatic nucleus (SCN) is the locus of a brain clock organizing circadian rhythmicity. In split hamsters, the right and left SCN oscillate 12 h out of phase with each other, and the twice-daily locomotor bouts alternately correspond to one or the other. This unique configuration of the circadian system is useful for investigation of SCN communication to efferent targets. To track phase and period in the SCN and its targets, we measured wheel-running and FOS expression in the brains of split and unsplit hamsters housed in LL or light-dark cycles. The amount and duration of activity before splitting were correlated with latency to split, suggesting behavioral feedback to circadian organization. LL induced a robust rhythm in the SCN core, regardless of splitting. The split hamsters' SCN exhibited 24-h rhythms of FOS that cycled in antiphase between left and right sides and between core and shell subregions. In contrast, the medial preoptic area, paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus and orexin-A neurons all exhibited 12-h rhythms of FOS expression, in-phase between hemispheres, with some detectable right-left differences in amplitude. Importantly, in all conditions studied, the onset of FOS expression in targets occurred at a common phase reference point of the SCN oscillation, suggesting that each SCN may signal these targets once daily. Finally, the transduction of 24-h SCN rhythms to 12-h extra-SCN rhythms indicates that each SCN signals both ipsilateral and contralateral targets.

Light intensity is an important determinant of diverse physiological and behavioral responses within the non-image-forming visual system. Thresholds differ among various photic responses, namely control of circadian rhythms, vigilance state, activity level and pupil constriction, but the mechanisms that regulate photosensitivity are not known. Calbindin D(28k) (CalB) is a calcium-binding protein associated with light processing in the mammalian circadian clock. Loss-of-function studies indicate that CalB-deficient mice (CalB(-/-)) have deficits in their ability to entrain to light-dark cycles. To explore the role of CalB in modulating photosensitivity, thresholds for three behaviors mediated by the non-image-forming visual system (entrainment, masking and pupillary light reflex; PLR) were compared in CalB(-/-) and wildtype mice, and the localization of CalB protein in these circuits was examined in adult and juvenile mice. The results reveal a divergence in how CalB affects thresholds to photic cues among these responses. Entrainment and masking were 40- to 60-fold less sensitive in CalB(-/-) than in wildtype mice. On the other hand, the PLR in CalB(-/-) mice was 80- to 200-fold more sensitive. Though CalB is expressed in the retina and in brain circuits regulating entrainment we found no CalB expression in any component of the PLR pathway, namely the olivary pretectal nucleus, Edinger-Westphal nucleus and ciliary ganglion. The behavioral and anatomical data together suggest that, in normal animals, the retinal response to light is blunted in the presence of CalB, but responsiveness of the higher order processes that transduce afferent retinal input is enhanced.

To conserve scarce energetic resources during winter, seasonal breeders inhibit reproduction and other nonessential behavioral and physiological processes. Reproductive cessation is initiated in response to declining day lengths, a stimulus represented centrally as a long-duration melatonin signal. The melatonin signal is not decoded by the reproductive axis directly, but by an unidentified neurochemical system upstream of gonadotropin-releasing hormone (GnRH). The dorsomedial nucleus of the hypothalamus (DMH) has been implicated in seasonal changes in reproductive function in Syrian hamsters ( Mesocricetus auratus), although the specific-cell phenotype decoding photoperiodic information remains unknown. RFamide-related peptide (RFRP; the mammalian homolog of the gonadotropin-inhibitory hormone (GnIH) gene identified in birds) has emerged as a potent inhibitory regulator of the reproductive axis and, significantly, its expression is localized to cell bodies of the DMH in rodents. In the present study, the authors explored the relationship between RFRP expression, photoperiod exposure, and reproductive condition/hormonal status. In male hamsters that respond to short days with reproductive inhibition, RFRP-ir and mRNA expression are markedly reduced relative to long-day animals. Replacement of testosterone in short-day animals did not affect this response, suggesting that alterations in RFRP expression are not a result of changing sex steroid concentrations. A subset of the hamster population that ignores day length cues and remains reproductively competent in short days (nonresponders) exhibits RFRP-ir expression comparable to long-day hamsters. Analysis of cell body and fiber density suggests a potential interplay between peptide production and release rate in differentially regulating the reproductive axis during early and late stages of reproductive regression. Together, the present findings indicate that photoperiod-induced suppression of reproduction is associated with changes in RFRP and mRNA expression, providing opportunity for further exploration on the role that RFRP plays in this process.

This paper reports a provocative and novel observation. Specifically, we have shown that in ring doves, following a brief period of courtship, cells of the immune system - mast cells- appear in the brain. This is surprising in that it has long been thought that the blood brain-barrier prevents the movement of blood-borne cells into the parenchyma of the brain. The conditions under which these cells appear, their chemical and ultrastructural features and their potential in neuroendocrine signalling functions are discussed.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D28K (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB) ⁄ ) mice). In constant darkness (DD), CalB) ⁄ ) animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB) ⁄ ) mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB) ⁄ ) mice suggest an important role for CalB in mouse circadian function.

In mammals, it is well established that circadian rhythms in physiology and behavior, including the rhythmic secretion of hormones, are regulated by a brain clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. While SCN regulation of gonadal hormone secretion has been amply studied, the mechanisms whereby steroid hormones affect circadian functions are less well known. This is surprising considering substantial evidence that sex hormones affect many aspects of circadian responses, and that there are significant sex differences in rhythmicity. Our previous finding that “core” and “shell” regions of the SCN differ in their expression of clock genes prompted us to examine the possibility that steroid receptors are localized to a specific compartment of the brain clock, with the discovery that the androgen receptor (AR) is concentrated in the SCN core in male mice. In the present study, we compare AR expression in female and male mice using Western blots and immunochemistry. Both of these methods indicate that AR’s are more highly expressed in males than in females; gonadectomy eliminates and androgen treatment restores these sex differences. At the behavioral level, gonadectomy produces a dramatic loss of the evening activity onset bout in males, but has no such effect in females. Treatment with testosterone, or with the non-aromatizable androgen, dihydrotestosterone restores male locomotor activity and eliminates sex differences in the behavioral response. The results indicate that androgenic hormones regulate circadian responses, and suggest an SCN site of action.

In nature and under laboratory conditions, dams nurse rabbit pups once daily for a duration of fewer than 5 min. The present study explored neural mechanisms mediating the timing of nursing in this natural model of food anticipatory activity, focussing on the suprachiasmatic nucleus (SCN), the locus of the master circadian clock and on the dorsomedial hypothalamic nucleus (DMH), a region implicated in timing of food-entrained behavior. Rabbit pups are born in the dark, with eyelids closed. Nursing visits to the litters also occurs during the dark phase. To explore the effect of the timing of feeding, pups were maintained in constant darkness, while females housed in a light-dark cycle were permitted to nurse their pups either during the night (night-fed group) or day (day-fed group). All pups exhibited anticipatory locomotor activity before daily nursing. In the SCN, PER1 and FOS peaked during the night in both groups, with a longer duration of elevated protein expression in the night-fed group. In contrast, DMH peak PER1 expression occurred 8 h after pups were fed, corresponding to the shift in timing of nursing. Comparison of nursed and 48 h fasted pups indicates that the timing of PER1 expression was similar in the SCN and DMH, with fewer PER1-positive cells in the latter group. The results indicate that rabbit pups show food anticipatory activity, and that timing of nursing differentially affects PER1 expression in the SCN and DMH.

The goal of the present study was to elucidate the role of DARPP-32 (dopamine- and cyclic adenosine 3'-5'-monophosphate-regulated phosphoprotein, 32 kDa) in retinal function. We examined mouse and rat retinas for the presence of DARPP-32 by immunocytochemistry. In both rodent retinas DARPP-32 immunoreactivity was localized to horizontal and AII amacrine neurons and to the Mueller glial cells, using immuno-double labelling. Additional unidentified neurons in the amacrine cell layer also showed DARPP-32 immunoreactivity. Using mice entrained to a 12-12 h light-dark cycle, we found that exposure to light presented during the dark phase significantly enhanced phosphorylation of DARPP-32 at threonine (Thr) 34 and phosphorylation of the ionotropic glutamate receptor subunit GluR1 at serine (Ser) 845, as measured by immunoblots. However, light also increased Ser 845-GluR1 phosphorylation in DARPP-32-knockout mice. When a dopamine D1 receptor antagonist was injected into the eye prior to light exposure, phosphorylation of both Thr 34-DARPP-32 and Ser 845-GluR1 was significantly reduced. These data indicate that DARPP-32 participates in dopamine-mediated modifications of retinal function. We also tested for a possible circadian rhythm of Thr 34- and Thr 75-DARPP-32 and Ser 845-GluR1 expression. No significant circadian rhythm of either DARPP-32 or GluR1 phosphorylation was found.

Suprachiasmatic nucleus (SCN) neuroanatomy has been a subject of intense interest since the discovery of the SCN's function as a brain clock and subsequent studies revealing substantial heterogeneity of its component neurons. Understanding the network organization of the SCN has become increasingly relevant in the context of studies showing that its functional circuitry, evident in the spatial and temporal expression of clock genes, can be reorganized by inputs from the internal and external environment. Although multiple mechanisms have been proposed for coupling among SCN neurons, relatively little is known of the precise pattern of SCN circuitry. To explore SCN networks, we examine responses of the SCN to various photic conditions, using in vivo and in vitro studies with associated mathematical modeling to study spatiotemporal changes in SCN activity. We find an orderly and reproducible spatiotemporal pattern of oscillatory gene expression in the SCN, which requires the presence of the ventrolateral core region. Without the SCN core region, behavioral rhythmicity is abolished in vivo, whereas low-amplitude rhythmicity can be detected in SCN slices in vitro, but with loss of normal topographic organization. These studies reveal SCN circuit properties required to signal daily time.

Hormone secretion is highly organized temporally, achieving optimal biological functioning and health. The master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus coordinates the timing of circadian rhythms, including daily control of hormone secretion. In the brain, the SCN drives hormone secretion. In some instances, SCN neurons make direct synaptic connections with neurosecretory neurons. In other instances, SCN signals set the phase of “clock genes” that regulate circadian function at the cellular level within neurosecretory cells. The protein products of these clock genes can also exert direct transcriptional control over neuroendocrine releasing factors. Clock genes and proteins are also expressed in peripheral endocrine organs providing additional modes of temporal control. Finally, the SCN signals endocrine glands via the autonomic nervous system, allowing for rapid regulation via multisynaptic pathways. Thus, the circadian system achieves temporal regulation of endocrine function by a combination of genetic, cellular, and neural regulatory mechanisms to ensure that each response occurs in its correct temporal niche. The availability of tools to assess the phase of molecular/cellular clocks and of powerful tract tracing methods to assess connections between “clock cells” and their targets provides an opportunity to examine circadian-controlled aspects of neurosecretion, in the search for general principles by which the endocrine system is organized.

Successful reproduction requires maintenance of the reproductive axis within fine operating limits through negative feedback actions of sex steroids. Despite the importance of this homeostatic process, our understanding of the neural loci, pathways, and neurochemicals responsible remain incomplete. Here, we reveal a neuropeptidergic pathway that directly links gonadal steroid actions to regulation of the reproductive system. An RFamide (Arg-Phe-NH 2 ) peptide that inhibits gonadotropin release from quail pituitary was recently identified and named gonadotropin-inhibitory hormone (GnIH). Birds are known to have specialized adaptations associated with gonadotropin-releasing hormone (GnRH) regulation to optimize reproduction (e.g., encephalic photoreceptors), and the existence of a hypothalamic peptide inhibiting gonadotropins may or may not be another such specialization. To determine whether GnIH serves as a signaling pathway for sex steroid regulation of the reproductive axis, we used immunohistochemistry and in situ hybridization to characterize the distribution and functional role of this peptide in hamsters, rats, and mice. GnIH-immunoreactive (GnIH-ir) cell bodies are clustered in the mediobasal hypothalamus with pronounced projections and terminals throughout the CNS. In vivo GnIH administration rapidly inhibits luteinizing hormone secretion. Additionally, GnIH-ir neurons form close appositions with GnRH cells, suggesting a direct means of GnRH modulation. Finally, GnIH-ir cells express estrogen receptor-α and exhibit robust immediate early gene expression after gonadal hormone stimulation. Taken together, the distribution of GnIH efferents to neural sites regulating reproductive behavior and neuroendocrine secretions, expression of steroid receptors in GnIH-ir nuclei, and GnIH inhibition of luteinizing hormone secretion indicate the discovery of a system regulating the mammalian reproductive axis.

We investigated parvalbumin immunoreactivity (PA-IR) in the retinas of rats maintained on a 12:12 h light:dark cycle, or after being placed in constant darkness for 24–72 h. Retinas were harvested at zeitgeber and circadian times 02:00, 06:00, 10:00, 14:00, 18:00 and 22:00 h. PA-IR was found primarily in retinal amacrine cells of the AII subtype. In a light/dark cycle, PA-IR showed a clear rhythm, with a low near zeitgeber time (ZT) 10:00 h and a peak near ZT 18:00 h. The ratio of immunofluorescence intensities at these timepoints was >15-fold. When animals were kept in complete darkness for 1–3 days, the rhythm of PA-IR was still preserved, but was progressively reduced in amplitude. The rhythm of PA-IR inferred from immunohistochemical data was confirmed by Western blots. We conclude that PA-IR in the rat retina shows an underlying circadian rhythm that is enhanced by cyclic light. The regulation may involve translocation of the protein between cell compartments and/or new protein synthesis.

Daily scheduled feeding is a potent time cue that elicits anticipatory activity in rodents. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from light-entrained oscillators of the suprachiasmatic nucleus (SCN). Circadian rhythms within the SCN depend on transcription-translation feedback loops in which CLOCK protein is a key positive regulator. The Clock gene is expressed in rhythmic tissues throughout the brain and periphery, implicating its widespread involvement in the functioning of circadian oscillators. To examine whether CLOCK protein is also necessary for the FEO, the effect of daily food restriction was studied in homozygous Clock mutant ( Clk/Clk) mice. The results show that Clk/Clk mutant mice exhibit FAA, even when their circadian wheel-running behavior is arrhythmic. As in wild-type controls, FAA in Clk/Clk mutants persists after temporal feeding cues are removed for several cycles, indicating that the FEO is a circadian timer. This is the first demonstration that the Clock gene is not necessary for the expression of a circadian, food-entrained behavior and suggests that the FEO is mediated by a molecular mechanism distinct from that of the SCN.

Maternal behaviour in the rabbit is unusual among mammals because the doe visits her litter to nurse once every 24 h. In the present study we examined the consequences of milk intake on oxytocinergic (OT) and vasopressinergic (AVP) neurons of the supraoptic (SON) and paraventricular (PVN) nuclei of 7-day-old pups before suckling, after suckling and following anogenital stroking in un-nursed pups. To determine neuronal activation we assessed the expression of the Fos protein combined with antibodies against OT and AVP at two levels in the SON (supraoptic rostral, SOr, and supraoptic retrochiasmatic, SOrch), and three levels in the PVN (anterior, PVab; medial PVm and caudal, PVc). Daily nursing bouts lasted only 228+/-6 s throughout the observed 7 days, and pups ingested up to 34.95+/-9.0% of their body weight in milk on day 7, the day of perfusion. Suckling induced a significant increase in the number of double-labeled Fos/OT cells in both subdivisions of the SON (P0.01) and in PVab and PVm (P0.01). The effect in the SON was related to suckling, as it was not seen in stroked, un-nursed pups, which showed Fos increases only in PVab and PVm. All regions in the SON and PVN showed significant increases in the number of Fos/AVP neurons after suckling or stroking but, contrary to OT, the number of double-labeled Fos/AVP cells was very low. In conclusion, our results show that the oxytocinergic system of the SON and PVN is differentially activated by suckling of milk and anogenital stroking, and that the vagal-hypothalamic axis is mature in 7-day-old rabbits.

Mast cells occur in the brain and their number changes with reproductive status. While it has been suggested that brain mast cells contain the mammalian hypothalamic form of gonadotropin-releasing hormone (GnRH-I), it is not known whether mast cells synthesize GnRH-I de novo. In the present study, mast cells in the rat thalamus were immunoreactive to antisera generated against GnRH-I and the GnRH-I associated peptide (GAP); mast cell identity was confirmed by the presence of heparin, a molecule specific to mast cells, or serotonin. To test whether mast cells synthesize GnRH-I mRNA, in situ hybridization was performed using a GnRH-I cRNA probe, and the signal was identified as being within mast cells by the binding of avidin to heparin. GnRH-I mRNA was also found, using RT-PCR, in mast cells isolated from the peritoneal cavity. Given the function of GnRH-I in the regulation of reproduction, changes in the population of brain GnRH-I mast cells were investigated. While housing males with sexually receptive females for 2 h or 5 days resulted in a significant increase in the number of brain mast cells, the proportion of mast cells positive for GnRH-I was similar to that in males housed with a familiar male. These findings represent the first report showing that mast cells synthesize GnRH-I and that the mast cell increase seen in a reproductive context is the result of a parallel increase in GnRH-I positive and non-GnRH-I positive mast cells.

Successful reproduction requires precise temporal coordination among various endocrine and behavioural events. The circadian system regulates daily temporal organization in behaviour and physiology, including neuroendocrine rhythms. The main circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sends direct efferents to the reproductive axis via monosynaptic projections to gonadotropin-releasing hormone (GnRH) neurones. This communication generates circadian endocrine rhythms as well as the preovulatory luteinizing hormone (LH) surge necessary for successful ovulation. One SCN peptide thought to be important for the regulation of oestrous cycles is vasoactive intestinal polypeptide (VIP). VIP neurones from the SCN contact GnRH cells, and these cells are preferentially activated during an LH surge in rats. Unlike adult rats, prepubertal females do not exhibit oestrous cycles, nor do they exhibit an LH surge in response to oestradiol positive-feedback. The present study was undertaken to determine the extent to which the development of a 'mature' reproductive axis in female rats is associated with modifications in VIP contacts on GnRH neurones. The brains of diestrus adult (approximately 60 days of age) and prepubertal (21 days of age) female rats were examined using double-label fluorescence immunohistochemistry for VIP and GnRH, with light and confocal microscopy. Although the total number of GnRH-immunoreactive neurones did not differ between adult and prepubertal females, adults had a significant increase in the percentage of GnRH cells receiving VIP contacts compared to juveniles. These data suggest that the development of reproductive hormone rhythms and oestrous cyclicity may be, in part, due to modifications of VIP input to the GnRH system.

Calbindin-D 28K-immunoreactive cells are tightly packed within a discrete region of the caudal aspect of the suprachiasmatic nuclei of hamsters. These cells receive direct retinal input and are Fos-positive in response to a light pulse. Knowledge of their aierent and eierent connections is necessary to understand suprachiasmatic nucleus organi- zation. The ¢rst aim of the present study is to identify interconnections between calbindin and other peptidergic cells of the suprachiasmatic nuclei, using epi- and confocal microscopy and intra-suprachiasmatic nucleus tract tracing. The results indicate that essentially all calbindin cells receive numerous appositions from vasoactive intestinal polypeptide (VIP), neuropeptide Y and serotonin ¢bers and that most receive appositions from gastrin releasing peptide (GRP) and cholecystokinin (CCK) ¢bers. Reciprocal connections are seen from VIP, GRP and CCK cells but surprisingly, not from dorsomedial vasopressin cells. Injection of biotinylated dextran amine into the suprachiasmatic nucleus indicates that the ventrolateral suprachiasmatic nucleus projects to the entire nucleus, while the dorsal and medial regions of the supra- chiasmatic nucleus project densely to most of the nucleus, except to the calbindin region. Analysis of colocalization of the peptides in the calbindin cell region shows that 91% of thesubstanceP ce lls, 42 % of theGRP ce lls and 60 % of theVIP cells in the calbindin subnucleus coexpress calbindin-D28K. Our results reveal a highly specialized topographical organization of connections among suprachiasmatic nucleus cells. C 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

We have explored the use of biotinylated dextran amine (BDA) as a marker for labeling fetal brain grafts and their connections with the host. As a model system we used transplantation of the hamster suprachiasmatic nucleus, the site of an endogenous biological clock governing circadian rhythms. Similar transplants into arrhythmic hosts have been shown to restore behavioral function with a period specific to the donor. For locomotor rhythms, efferent connections are not necessary. For other responses, including endocrine rhythms, efferent connections may be necessary. In order to visualize homografts and their efferents, injections of BDA, an anterograde tracer, were made into the anterior hypothalamic (AH) region containing the SCN or into the dorsal cortex (CTX) of fetal hamster brains. The fetal AH or CTX was microdissected out and stereotaxically implanted into the third ventricle of intact, adult hamsters. After 2, 4, 8, or 12 weeks, hosts were sacrificed and their brains were processed for detection of BDA by either histochemistry or immunofluorescence. BDA intensely labeled graft neurons, their dendrites, and axons with minimal or no spread to the adjacent host brain. Labeled graft axons could be followed for long distances (>1 mm) into the host brain and graft-derived varicosities formed close contacts with host neurons. BDA-labeled graft neurons, located at the perimeter of the graft, also extended dendrite-like processes into the host parenchyma. We conclude that BDA is a useful marker for fetal homografts and their efferents for survival times of less than 2 months.

The mammalian circadian clock lying in the suprachiasmatic nucleus (SCN) controls daily rhythms and synchronizes the organism to its environment. In all organisms studied, circadian timekeeping is cell-autonomous, and rhythmicity is thought to be generated by a feedback loop involving clock proteins that inhibit transcription of their own genes. In the present study, we examined how these cellular properties are organized within the SCN tissue to produce rhythmicity and photic entrainment. The results show that the SCN has two compartments regulating Period genes Per1, Per2, and Per3 mRNA expression differentially. One compartment shows endogenous rhythmicity in Per1, Per2, and Per3 mRNA expression. The other compartment does not have rhythmic mRNA expression but has gated light-induced Per1 and Per2 and high levels of endogenous nonrhythmic Per3 mRNA expression. These results reveal the occurrence of differential regulation of clock genes in two distinct SCN regions and suggest a potential mechanism for producing functional differences in distinct SCN subregions.

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.

While mast cells in connective tissues have long been associated with allergic reactions, it is now clear that they are also present within the central nervous system under normal physiological conditions. The mast cell population increases 10-fold in the medial habenular region of the brain within 2 h after pairing in doves. The first study explored whether this increase was due to exposure to gonadal steroids. Light microscopic immunocytochemistry indicates an increased number of brain MC following exposure to either testosterone (T) or dihydrotestosterone (DHT) in the male, or 17β estradiol (E) in the female, but not in cholesterol-treated controls. Thus, the increased habenular MC population is produced by gonadal hormones in the absence of sexual behavior, is not sexually dimorphic, and does not require aromatization of androgen. In the next study, MC activational state was determined using electron microscopy. Cells were categorized into five states: (I) resting; (II) initiation of degranulation; (I...

Mast cells are found in the brain of many species. Although a considerable body of information is available concerning the development and differentiation of peripheral mast cells, little is known about brain mast cells. In the present study, the ontogeny of mast cells in the dove brain was followed by using three markers: acidic toluidine blue, alcian blue/safranin, and an antiserum to gonadotropin-releasing hormone (GnRH). Mast cells first appear in the pia on embryonic day (E)13-14 in ovo, then along blood vessels extending from the pia into the telencephalon on posthatch day 4-5, and in the medial habenula at week 3. Medial habenular mast cell numbers increase during development, peaking in peripubertal birds, and declining thereafter. Several measures indicate that mast cells mature within the medial habenula: there is an increase in the intensity of metachromasia, a switch from alcian blue granules in young animals to mixed alcian blue and safranin granules in older animals, and an increase in GnRH-like immunoreactivity. These results were extended by using electron microscopy. The architecture of mast cell granules evolved from electron lucent with small electron dense deposits at E15 to more electron dense granules with complex patterns of internal structure by 2 months. Ultrastructural immunocytochemistry for the GnRH-like peptide at 1 month revealed both immunopositive and negative cells, suggesting that the acquisition of this phenotype is not simultaneous across the population. Thus, immature mast cells infiltrate the central nervous system and undergo in situ differentiation within the neuropil.

Fetal grafts of the anterior hypothalamus (SCN/AH) containing the suprachiasmatic nucleus (SCN) restore circadian rhythms to SCN-lesioned host hamsters and rats following implantation into the third ventricle. Previous studies suggest that intraventricular SCN/AH grafts are variable in their attachment sites, the extent of their outgrowth, and the precise targets innervated in the host brain. However, the use of different methods to analyze graft outgrowth in this model has previously led to inconsistent results. We have reevaluated the outgrowth of fetal rat SCN/AH grafts implanted in the third ventricle of hamsters by using two methods: the carbocyanine dye, 1,1'dioctadecyl-3,3'-tetramethylindocarbocyanine percholate (DiI), was placed directly onto grafted tissue; and a donor-specific neurofilament marker was used in conjunction with xenografts. We examined the specificity of outgrowth by comparing SCN/AH xenografts with that of control cortical (CTX) xenografts. To evaluate whether SCN/AH graft efferents arise from the donor SCN, we used micropunch grafts that contained minimal extra-SCN tissue. The results show that the use of a donor-specific neurofilament marker reveals more extensive SCN/AH graft outgrowth than DiI. SCN/AH graft efferents project into areas normally innervated by the intact SCN. However, this outgrowth is variable among graft recipients, is not specific to SCN/AH tissue, and does not necessarily derive from the donor SCN. The precise functional role of neural efferents arising from SCN/AH grafts in the restoration of circadian clock function and the extent of SCN-derived efferents remain to be determined.

Fetal hypothalamic grafts containing the suprachiasmatic nucleus (SCN) restore circadian locomotor rhythmicity when implanted into the third ventricle of SCN-lesioned hamsters. However, the quality of restored rhythms is variable, and the locomotor rhythms of grafted animals are generally less robust than those of intact animals. The present study explored whether anatomical features of the graft predict the quality of the recovered rhythm and whether such information might provide insight as to the target of the signal from the SCN that controls locomotor rhythmicity. The following graft parameters were assessed: distance between the attachment site of the graft and potential targets for the output signal from the SCN, number and overall size of SCN clusters, the size of the cluster closest to the SCN lesion site, and extent of vasoactive intestinal polypeptide (VIP) and vasopressin-associated neurophysin (NP) positive fiber outgrowth from the graft. The restored circadian activity rhythm was assessed by quantifying the precision of activity onset and the amount, period, and robustness of rhythmicity. The results indicate a significant positive correlation between the precision of activity onset and the proximity of the closest SCN cluster to the site of the lesioned host SCN. A more detailed analysis of the spatial location of the graft indicates that proximity of the graft in the dorsal and caudal directions, but not the rostral direction, is positively correlated with the precision of the recovered rhythm. This suggests two possibilities: the coupling signal may act on a site very near the SCN and travel preferentially in a rostro-caudal direction. Alternatively, the coupling signal may act on a site rostral to the SCN. That the site is not far rostral to the SCN was suggested by the lack of a correlation between the precision of the restored rhythm and the rostrally lying anterior medial preoptic nucleus. Finally, evaluation of NP- and VIP-ergic fibers in nuclei known to receive input from the SCN indicates that the extent of such innervation by graft efferents does not predict either the occurrence of recovery or the precision of the recovered rhythm. Overall, these results suggest that the target(s) of SCN pacemakers regulating locomotor rhythmicity lie in the hypothalamus, close to or rostral to the SCN.

Mast cells synthesize vasoactive agents and a number of neurotransmitters. They are particularly numerous in the medial habenular region of the epithalamus, the attachment site of the choroid plexus. The present study examined whether degranulation of brain mast cells alters the permeability of the blood-brain barrier (BBB). To this end, doves were injected intramuscularly with the mast cell degranulator, compound 48/80 (C40/80), followed by i.v. injection of Evans blue. The distribution of the dye in the parenchyma was examined using digital imaging. Three brain areas were analyzed: the medial habenula (which also contains mast cells), the paraventricular nucleus (PVN, which abuts the third ventricle, but has no mast cells), and the lateral septal organ (LSO, a circumventricular organ with fenestrated capillaries). Significantly more Evans blue tracer and fewer toluidine blue-positive mast cells were detected in the medial habenula of subjects treated with C48/80 compared to saline controls. Evans blue did not enter the PVN in either the experimental or control group, while it entered the LSO equally in both. Degranulation of mast cells after C48/80 treatment was confirmed histochemically and ultrastructurally. The results support the hypothesis that brain mast cell degranulation locally alters BBB permeability. Activation of brain mast cells may provide a mechanism for regulated opening of the BBB.

The mammalian suprachiasmatic nuclei (SCN) transmit signals to the rest of the brain, organizing circadian rhythms throughout the body. Transplants of the SCN restore circadian activity rhythms to animals whose own SCN have been ablated. The nature of the coupling signal from the grafted SCN to the host brain is not known, although it has been presumed that functional recovery requires re-establishment of appropriate synaptic connections. We have isolated SCN tissue from hamsters within a semipermeable polymeric capsule before transplantation, thereby preventing neural outgrowth but allowing diffusion of humoral signals. Here we show that the transplanted SCN, like neural pacemakers of Drosophila and silkmoths, can sustain circadian activity rhythms by means of a diffusible signal.

Although it is widely accepted that the suprachiasmatic nuclei (SCN) of the hypothalamus serve as biological pacemakers regulating circadian rhythmicity, a number of studies suggest that some circadian rhythms may be controlled by extra-SCN structures. Transplantation of fetal anterior hypothalamic tissue containing the SCN restores circadian locomotor rhythms in SCN-lesioned hosts. Such transplants, however, contain substantial extra-SCN hypothalamic tissue. In the present study, the authors examined the recovery of circadian locomotor rhythms in animals implanted with small grafts harvested by taking "micropunches" from vibratome-sectioned brain slices. Micropunches were taken from three areas of the hypothalamus known to receive retinal input: the SCN, the subparaventricular zone, and the supraoptic nucleus. The results indicate that transplants restricted to the SCN region are necessary and sufficient for restoration of circadian locomotor activity rhythms and that micropunches of tissues from other sources are ineffective.

Mother rabbits construct an elaborate maternal nest before parturition and display a single, brief, daily nursing bout throughout lactation. These features present a unique model for investigating the relevance of changes in neuroendocrine secretion associated with pregnancy and parturition for the regulation of maternal behavior. In the present study we analyzed changes in the location, somal size, and number of oxytocin (OT)and arginine vasopressin (AVP)-immunoreactive (IR) neurons in the hypothalamus of rabbits in estrus, late pregnancy (day 29), and postpartum day 1. From estrus to late pregnancy, the number of OT-IR neurons increased in the scattered cell groups located in the lateral hypothalamic area (LHA), but not in the magnocellular nuclei, i.e., paraventricular nucleus (PVN) and supraoptic nucleus (SON). On postpartum day 1 the increase in the number of OT-IR neurons was sustained in the LHA and became apparent also in the main body of the PVN, in which the number of OT-IR neurons doubled. Increases in the somal size of OT-IR cells were seen in all three nuclei only on postpartum day 1. No OT-IR cells were found in the suprachiasmatic nucleus (SCN). From late pregnancy and into postpartum day 1 increases in the somal size of AVP-IR neurons were detected in the PVN, SON, and LHA but not in the SCN. The number of AVP-IR neurons increased between late pregnancy and postpartum day 1 in the SON only. The changes observed in OT and AVP expression in specific hypothalamic nuclei may be related to specific somatic and behavioral events occurring around the time of parturition, e.g., nest-building, maintenance of homeothermy, elevation of blood volume, and nursing in mother rabbits.

The hypothalamic suprachiasmatic nucleus (SCN) is the locus of a master clock that regulates circadian rhythms in physiology and behavior. Gonadectomy in male mice lengthens the period of circadian rhythms and increases the day-to-day variability of activity onset time. Both of these responses are rescued by the nonaromatizable androgen dihydrotestosterone. Androgen receptors (AR) are localized in SCN neurons that receive direct retinal input. To explore how androgens affect circadian clock function and its responsiveness to photic cues, we measured wheel-running behavior and SCN AR expression in intact, gonadectomized, and testosterone-replaced mice, held under various photic conditions. Gonadectomy lengthened circadian period in constant dim light but not in constant darkness. Increasing intensities of constant light parametrically increased circadian period, and this was potentiated at all intensities by gonadectomy. In contrast, gonadectomy did not alter light-induced pupil constriction, suggesting a nonretinal locus of hormone action. In hormone-replaced animals housed in constant darkness, T concentration was positively correlated with precision of activity onset and with SCN AR expression and negatively correlated with duration of activity. We infer the existence of two androgenic mechanisms: one modulates SCN responsiveness to light, and the second modulates SCN timekeeping and locomotor activity in a dose-dependent manner. Finally, the effects of androgens on period are a result of hormonal modulation of the SCN's response to photic input rather than to a change in the inherent period of oscillators in the absence of light.

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.

It is widely accepted that the suprachiasmatic nuclei (SCN) of the hypothalamus serve as biological pacemakers, organizing daily activities. However some circadian rhythms are controlled by extra-SCN structures. Transplantation of fetal donor SCN in SCN-lesioned rodents induces recovery of rhythmic locomotor and drinking activities. Such grafts do not however, restore appropriate gonadal responses to photoperiodic stimuli. It is not known whether other behavioral rhythms are restored by fetal tissue grafts, or whether various responses are restored simultaneously. In the present study, we established that circadian rhythms of gnawing behavior are abolished following SCN lesions. Next, we measured both gnawing and wheel-running activity in SCN-lesioned hamsters following transplantation of fetal hypothalamic grafts containing the SCN. The results indicate that such grafts restore circadian rhythms of gnawing behavior, and that gnawing and wheel-running rhythms re-emerge at about the same time.

Most temperate zone birds show dramatic seasonal cycles in responsiveness to light. In the spring the hypothalamo-pituitary-gonadal axis of photosensitive birds is stimulated by long days. In the late summer birds no longer respond to long days, their gonads regress, and they are said to be photorefractory. After several weeks of refractoriness birds regain photosensitivity. During refractoriness circulating concentrations of luteinizing hormone are low and prolactin levels are high. These fluctuations in peripheral hormones result from changes in the brain rather than in the pituitary and/or the gonads. In the present study we examined seasonal changes in expression of vasoactive-intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH) in the brain of dark-eyed juncos (Junco hyemalis). Birds were photosensitive and exposed to long photoperiod (20:4 LD) for 1 day, 45-60 days, or not at all, or they were photorefractory (housed in 20:4 LD). The results indicate that VIP expression was similar in all photosensitive birds. However, photorefractory birds had significantly higher numbers of VIP-positive neurons in the infundibulum compared to photosensitive birds. The number of GnRH-positive neurons in the preoptic area was significantly lower in photorefractory birds and significantly higher in long-term photostimulated birds. These results indicate that the inverse relationship between circulating prolactin (released by VIP) and luteinizing hormone (released by GnRH) during refractoriness may result from neural changes in VIP and GnRH expression, respectively.

While much is known about the mechanisms that underlie sleep and circadian rhythms, the investigation into sex differences and gonadal steroid modulation of sleep and biological rhythms is in its infancy. There is a growing recognition of sex disparities in sleep and rhythm disorders. Understanding how neuroendocrine mediators and sex differences influence sleep and biological rhythms is central to advancing our understanding of sleep-related disorders. While it is known that ovarian steroids affect circadian rhythms in rodents, the role of androgen is less understood. Surprising findings that androgens, acting via androgen receptors in the master “circadian clock” within the suprachiasmatic nucleus, modulate photic effects on activity in males point to novel mechanisms of circadian control. Work in aromatase-deficient mice suggests that some sex differences in photic responsiveness are independent of gonadal hormone effects during development. In parallel, aspects of sex differences in sleep are also reported to be independent of gonadal steroids and may involve sex chromosome complement. This a summary of recent work illustrating how sex differences and gonadal hormones influence sleep and circadian rhythms that was presented at a Mini-Symposium at the 2011 annual meeting of the Society for Neuroscience.

Huntington's disease (HD) is a progressive neurodegenerative disease marked by psychiatric and motor problems. Recently, these findings have been extended to deficits in sleep and circadian function that can be observed in HD patients and in HD mouse models, with abnormal sleep patterns correlating with symptom severity in patients. Here, we studied the behavior of the BAC HD mouse model using an 24/7 automated system; the results indicate significant lengthening of the circadian period in the mutant mice. These results reinforce previous findings in HD models and symptomatic HD patients, indicating that circadian dysfunction is a core feature of HD. Abstract Huntington's disease (HD) is a progressive neurodegenerative disease marked by psychiatric and motor problems. Recently, these findings have been extended to deficits in sleep and circadian function that can be observed in HD patients and in HD mouse models, with abnormal sleep patterns correlating with symptom severity in patients. Here, we studied the behavior of the BAC HD mouse model using an 24/7 automated system; the results indicate significant lengthening of the circadian period in the mutant mice. These results reinforce previous findings in HD models and symptomatic HD patients, indicating that circadian dysfunction is a core feature of HD. Abstract Huntington's disease (HD) is a progressive neurodegenerative disease marked by psychiatric and motor problems. Recently, these findings have been extended to deficits in sleep and circadian function that can be observed in HD patients and in HD mouse models, with abnormal sleep patterns correlating with symptom severity in patients. Here, we studied the behavior of the BAC HD mouse model using an 24/7 automated system; the results indicate significant lengthening of the circadian period in the mutant mice. These results reinforce previous findings in HD models and symptomatic HD patients, indicating that circadian dysfunction is a core feature of HD.

Gonadal hormones can modulate circadian rhythms in rodents and humans, and androgen receptors are highly localized within the core region of the mouse suprachiasmatic nucleus (SCN) brain clock. Although androgens are known to modulate neural plasticity in other CNS compartments, the role of androgens and their receptors on plasticity in the SCN is unexplored. In the present study, we ask whether androgens influence the structure and function of the mouse SCN by examining the effects of gonadectomy (GDX) on the structure of the SCN circuit and its responses to light, including induction of clock genes and behavioral phase shifting. We found that after GDX, glial fibrillary acidic protein increased with concomitant decreases in the expression of the synaptic proteins synaptophysin and postsynaptic density 95. We also found that GDX exerts effects on the molecular and behavioral responses to light that are phase dependent. In late night [circadian time (CT)21], GDX increased light-induced mPer1 but not mPer2 expression compared with intact (INT) controls. In contrast, in early night (CT13.5), GDX decreased light induced mPer2 but had no effect on mPer1. At CT13.5, GDX animals also showed larger phase delays than did INT. Treatment of GDX animals with the nonaromatizable androgen dihydrotestosterone restored glial fibrillary acidic protein, postsynaptic density 95, and synaptophysin in the SCN and reinstated the INT pattern of molecular and behavioral responses to light. Together, the results reveal a role for androgens in regulating circuitry in the mouse SCN, with functional consequences for clock gene expression and behavioral responses to photic phase resetting stimuli.

Heavy water (D 2 O) lengthens the period of free-running circadian rhythms in most organisms. We compared the effect of D 2 O on freerunning locomotor activity rhythms in intact and SCN-lesioned (SCN-X) hamsters that had recovered circadian rhythmicity following implantation of SCN grafts. The animals were housed individually in cages equipped with running wheels, and locomotor activity was monitored using a computer-based data acquisition system. At the end of the behavioral tests, animals were anesthetized and perfused. Brain sections were immunostained for vasoactive intestinal peptide (VIP) and vasopressin (VP) to evaluate the extent of the lesion and the presence of a functional graft. The D 2 O similarly lengthened the period of free-running activity without affecting amount of activity in both intact and in SCN-X grafted animals. The results indicate that D 2 O acts directly on the SCN to lengthen the free-running period, and suggest that coupling between pacemakers within the grafted SCN is as efficient as in the intact SCN.

Male and female ring doves express a sexually dimorphic pattern of incubation. The dimorphism is temporal rather than motoric. The male incubates for a block of time in the middle of the day and the female incubates the rest of the time. The present study explored the role of gonadal hormones in the control of the temporal dimorphism. Female-female pairs incubated their eggs, but it could not be predicted which of the partners would be sitting on the nest at any given time. Male-male pairs did not incubate and instead destroyed the nests that were provided and displayed aggressive behavior. Some intact males incubated when paired with gonadectomized males, although the castrates tended to ignore the nest. In contrast, when castrated birds were given heterotypical hormonal replacement therapy, they and their same-sex partners incubated the eggs that had been provided, with the gonadectomized birds sitting at a time appropriate to the hormonal state. The results indicate that gonadal hormones influence not only the expression of incubation behavior, but also its phase and duration.

Calbindin-D28K (CalB)—containing cells form a distinct cluster within the core of the hamster suprachiasmatic nucleus (SCN). These cells are directly retinorecipient but lack detectable rhythms in clock gene expression or electrical activity. In studies exploring SCN connectivity using double-label immunochemistry, we previously reported an absence of contacts among CalB fibers and vasopressin (VP) cells in animals sacrificed during the day. Here, we explored circadian variations in CalB-immunoreactivity (-ir) and re-examined the connections between CalB and other SCN cell types at zeitgeber times (ZT) 4 and 14. The results reveal a circadian rhythm of CalB-ir in fibers of SCN cells with high expression during the night and subjective night and low expression during the day and subjective day. This circadian difference is not seen in the other brain regions studied. Significantly more appositions were detected between CalB fibers and VP cells during the night than during the day, while circadian variation in numbers of contacts was not seen between CalB fibers and vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), or gastrin-releasing peptide (GRP) cells. There was no detectable variation in appositions from any peptidergic fiber type onto CalB cells. The present findings suggest that CalB cells relay photic information to VP oscillator cells of the SCN shell in a temporally gated manner.

Increases in arousal and activity in anticipation of a meal, termed “food anticipatory activity” (FAA), depend on circadian food-entrainable oscillators (FEOs), whose locations and output signals have long been sought. It is known that ghrelin is secreted in anticipation of a regularly scheduled mealtime. We show here that ghrelin administration increases locomotor activity in nondeprived animals in the absence of food. In mice lacking ghrelin receptors, FAA is significantly reduced. Impressively, the cumulative rise of activity before food presentation closely approximates a Gaussian function ( r = 0.99) for both wild-type and ghrelin receptor knockout animals, with the latter having a smaller amplitude. For both groups, once an animal begins its daily anticipatory bout, it keeps running until the usual time of food availability, indicating that ghrelin affects response threshold. Oxyntic cells coexpress ghrelin and the circadian clock proteins PER1 and PER2. The expression of PER1, PER2, and ghrelin is rhythmic in light–dark cycles and in constant darkness with ad libitum food and after 48 h of food deprivation. In behaviorally arrhythmic-clock mutant mice, unlike control animals, there is no evidence of a premeal decrease in oxyntic cell ghrelin. Rhythmic ghrelin and PER expression are synchronized to prior feeding, and not to photic schedules. We conclude that oxyntic gland cells of the stomach contain FEOs, which produce a timed ghrelin output signal that acts widely at both brain and peripheral sites. It is likely that other FEOs also produce humoral signals that modulate FAA.

The prominent circadian rhythms of the endocrine system are important for our broader understanding of physiology and behavior, in both health and disease. There is now much evidence that disruptions in the circadian domain, either due to external factors such as shift work or internal factors such as sleep disturbances, can lead to physiological and psychological pathologies. There is interest and urgency in understanding how time is coordinated in the body. A master circadian clock resides in the suprachiasmatic nucleus of the hypothalamus. While this is the only tissue in which autonomous rhythms are self-sustaining, the molecular machinery is present in most peripheral cells. Rather than imposing time on the body then, the SCN entrains local clocks in a tissue-specific manner. These peripheral clocks then participate in controlling rhythmic transcription of genes associated with local tissue function. In this review, we examine the interplay between brain and peripheral clocks in the control of endocrine rhythms.

The suprachiasmatic nuclei (SCN) regulate circadian rhythmicity in many biological and behavioral responses. Hamsters are made permanently arrhythmic by bilateral destruction of the SCN. Circadian locomotor rhythmicity is restored by fetal tissue transplants placed in the 3rd ventricle (3V). If intact animals are implanted with fetal SCN grafts, they maintain locomotor activity rhythms when the host SCN are subsequently destroyed. The mechanism(s) whereby the SCN (either grafted or in situ) regulate locomotor rhythmicity is not known. Evidence from other graft models point to the possibility of efferents to appropriate targets in the host. In the present study, efferent connections of transplanted fetal SCN were examined using the carbocyanine dye, Dil. Intact or SCN-lesioned animals were sacrificed 7 or 40 days after receiving fetal SCN grafts into 3V. Dil crystals were placed on the grafts in fixed brains which were then incubated for 3-6 weeks before sectioning. Sections bearing Dil-labelled efferents from the graft were photographed and then stained for immunoreactive VIP and NP cells to locate donor SCN. Although labelled efferents were observed in a majority of the grafts, most were confined to the limits of the graft. The few labelled efferents that entered the host tissue when the graft seemed to merge with the host did not extend very far regardless of whether the graft contained immunohistochemical evidence for donor SCN or not. The observation of limited graft-host connectivity suggests either that a limited number of efferents is sufficient to support circadian locomotor rhythmicity, or that the mechanism mediating restoration of function entails a diffusible substance.

Surgical isolation of the suprachiasmatic nuclei (SCN) within a hypothalamic island is reported to produce loss of circadian rhythmicity. The results have been interpreted to indicate that SCN efferents are necessary for the expression of circadian rhythms. It is not clear, however, whether the loss of circadian rhythms in behavioral responses following SCN isolation is attributable to transection of efferents, to loss of cells within the island, or to gliosis produced by the knife cut. To explore this issue, we examined locomotor activity and gonadal state of male golden hamsters housed in constant darkness (DD, with a dim red light for maintenance) for at least 10 weeks following isolation of the SCN from the rest of the brain by cuts by means of a Halasz wire microknife. Brain sections were immunocytochemically stained for the peptides vasoactive intestinal polypeptide (VIP), vasopressin (VP) or neurophysin II (NP II), and neuropeptide Y (NPY) to localize the SCN and to assess its viability, and for glial fibrillary acidic protein (GFAP) to delimit the border of the knife cut. Experimental animals with VIP and VP/NP II immunoreactivity in the SCN within the island retained free-running locomotor rhythms following transection of SCN efferents. Animals with cuts that failed to sever SCN efferents, and sham-operated animals (in which the Halasz knife was lowered but not rotated), also maintained circadian rhythmicity. Hamsters sustaining severe damage to the SCN showed disrupted locomotor activity. In those hamsters that retained circadian locomotor rhythmicity following SCN isolation, gonads failed to regress in DD, demonstrating the absence of an appropriate photoperiodic response. The results suggest a multiplicity of SCN coupling mechanisms in the control of circadian rhythms.

As central nervous system residents, mast cells contain many cytokines and are localized primarily near large blood vessels in the diencephalon and within the leptomeninges, making them candidates for immune to neural “cross talk.” Using mast cell-deficient KitW-sh/W-sh mice, we assessed the role of these cells in the thermoregulatory component of the immune response to lipopolysaccharide (LPS). KitW-sh/W-sh and wild-type (WT) mice differed in several respects in response to injection of a high dose of LPS (1 mg/kg ip). Core temperature (Tc) of WT mice decreased by ∼3°C, whereas KitW-sh/W-sh mice did not become hypothermic but instead exhibited pronounced low-frequency Tc oscillations around their baseline temperature. In addition, KitW-sh/W-sh mice had lower levels of whole brain TNF-α but no differences in IL-1β, IL-6, IFN-γ, or histamine compared with WT mice following injection of the high dose of LPS, consistent with the role of TNF-α in sepsis. KitW-sh/W-sh mice had increased resistance to LPS, and some survived a dose of LPS that was lethal in littermate controls. In contrast, KitW-sh/W-sh and WT mice were similar in other aspects, namely, in the hyperthermia following injection of TNF-α (1.5 μg icv), reduced nighttime Tc and locomotor activity (to 1 mg/kg LPS), response to a low dose of LPS (10 μg/kg ip), and response to subcutaneous turpentine injection. These results indicate that mast cells play a role in the regulation of thermoregulatory responses and survival following sepsis induction and suggest a brain site of action.

The circadian clock in the suprachiasmatic nucleus (SCN) plays a critical role in seasonal processes by sensing ambient photoperiod. To explore how it measures day-length, we assessed the state of SCN oscillators using markers for neuronal activity (c-FOS) and the clock protein (PER1) in Syrian hamsters housed in long (LD, 16 : 8 h light : dark) vs. short days (SD, 8 : 16 h light : dark). During SD, there was no detectable phase dispersion across the rostrocaudal extent of the nucleus. In contrast, during LD, rhythms in the caudal SCN phase led those in the mid- and rostral SCN by 4-8 h and 8-12 h, respectively. Importantly, some neurons in the retinorecipient core SCN were unique in that they were FOS-positive during the dark phase in LD, but not SD. Transfer of LD animals to constant darkness or skeleton photoperiod revealed that dark-phase FOS expression depends on tonic light exposure rather than on intrinsic clock properties. By transferring animals from SD to LD, we next discovered that there are two separate populations of SCN cells, one responding to acute and the other to tonic light exposure. The results suggest that the seasonal encoding of day-length by the SCN entails reorganization of its constituent oscillators by a subgroup of neurons in the SCN core that respond to tonic photic cues.