Day-night differences in neural activation in histaminergic and serotonergic areas with putative projections to the cerebrospinal fluid in a diurnal brain.

In nocturnal rodents, brain areas that promote wakefulness have a circadian pattern of neural activation that mirrors the sleep/wake cycle, with more neural activation during the active phase than during the rest phase. To investigate whether differences in temporal patterns of neural activity in wake-promoting regions contribute to differences in daily patterns of wakefulness between nocturnal and diurnal species, we assessed Fos expression patterns in the tuberomammillary (TMM), supramammillary (SUM), and raphe nuclei of male grass rats maintained in a 12:12 h light-dark cycle. Day-night profiles of Fos expression were observed in the ventral and dorsal TMM, in the SUM, and in specific subpopulations of the raphe, including serotonergic cells, with higher Fos expression during the day than during the night. Next, to explore whether the cerebrospinal fluid is an avenue used by the TMM and raphe in the regulation of target areas, we injected the retrograde tracer cholera toxin subunit beta (CTB) into the ventricular system of male grass rats. While CTB labeling was scarce in the TMM and other hypothalamic areas including the suprachiasmatic nucleus, which contains the main circadian pacemaker, a dense cluster of CTB-positive neurons was evident in the caudal dorsal raphe, and the majority of these neurons appeared to be serotonergic. Since these findings are in agreement with reports for nocturnal rodents, our results suggest that the evolution of diurnality did not involve a change in the overall distribution of neuronal connections between systems that support wakefulness and their target areas, but produced a complete temporal reversal in the functioning of those systems.

Changes in and dorsal to the rat suprachiasmatic nucleus during early pregnancy.

Circadian rhythms in behavior and physiology change as female mammals transition from one reproductive state to another. The mechanisms responsible for this plasticity are poorly understood. The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian pacemaker in mammals, and a large portion of its efferent projections terminate in the ventral subparaventricular zone (vSPZ), which also plays important roles in rhythm regulation. To determine whether these regions might mediate changes in overt rhythms during early pregnancy, we first compared rhythms in Fos and Per2 protein expression in the SCN and vSPZ of diestrous and early pregnant rats maintained in a 12:12-h light/dark (LD) cycle. No differences in the Fos rhythm were seen in the SCN core, but in the SCN shell, elevated Fos expression was maintained throughout the light phase in pregnant, but not diestrous, rats. In the vSPZ, the Fos rhythm was bimodal in diestrous rats, but this rhythm was lost in pregnant rats. Peak Per2 expression was phase-advanced by 4 h in the SCN of pregnant rats, and some differences in Per2 expression were found in the vSPZ as well. To determine whether differences in Fos expression were due to altered responsivity to light, we next characterized light-induced Fos expression in the SCN and vSPZ of pregnant and diestrous rats in the mid-subjective day and night. We found that the SCN core of the two groups responded in the same way at each time of day, whereas the rhythm of Fos responsivity in the SCN shell and vSPZ differed between diestrous and pregnant rats. These results indicate that the SCN and vSPZ are functionally re-organized during early pregnancy, particularly in how they respond to the photic environment. These changes may contribute to changes in overt behavioral and physiological rhythms that occur at this time.

Neural activation in arousal and reward areas of the brain in day-active and night-active grass rats.

In the diurnal unstriped Nile grass rat (Arvicanthis niloticus) access to a running wheel can trigger a shift in active phase preference, with some individuals becoming night-active (NA), while others continue to be day-active (DA). To investigate the contributions of different neural systems to the support of this shift in locomotor activity, we investigated the association between chronotype and Fos expression during the day and night in three major nuclei in the basal forebrain (BF) cholinergic (ACh) arousal system - medial septum (MS), vertical and horizontal diagonal band of Broca (VDB and HDB respectively) -, and whether neural activation in these areas was related to neural activity in the orexinergic system. We also measured Fos expression in dopaminergic and non-dopaminergic cells of two components of the reward system that also participate in arousal - the ventral tegmental area (VTA) and supramammillary nucleus (SUM). NAs and DAs were compared to animals with no wheels. NAs had elevated Fos expression at night in ACh cells, but only in the HDB. In the non-cholinergic cells of the BF of NAs, enhanced nocturnal Fos expression was almost universally seen, but only associated with activation of the orexinergic system for the MS/VDB region. For some of the areas and cell types of the BF, the patterns of Fos expression of DAs appeared similar to those of NAs, but were never associated with activation of the orexinergic system. Also common to DAs and NAs was a general increase in Fos expression in non-dopaminergic cells of the SUM and anterior VTA. Thus, in this diurnal species, voluntary exercise and a shift to a nocturnal chronotype changes neural activity in arousal and reward areas of the brain known to regulate a broad range of neural functions and behaviors, which may be also affected in human shift workers.

Rhythmic cFos expression in the ventral subparaventricular zone influences general activity rhythms in the Nile grass rat, Arvicanthis niloticus.

Circadian rhythms in behavior and physiology are very different in diurnal and nocturnal rodents. A pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus is responsible for generating and maintaining circadian rhythms in mammals, and cellular and molecular rhythms within the SCN of diurnal and nocturnal rodents are very similar. The neural substrates determining whether an animal has a diurnal or nocturnal phase preference are thus likely to reside downstream of the SCN. The ventral subparaventricular zone (vSPVZ), a major target of the SCN that is important for the expression of circadian rhythmicity in nocturnal lab rats (Rattus norvegicus), exhibits different rhythms in cFos expression in diurnal Nile grass rats compared to lab rats. We examined the effects of chemotoxic lesions of the cFos-expressing cells of the vSPVZ on activity rhythms of grass rats to evaluate the hypothesis that these cells support diurnality in this species. Male grass rats housed in a 12:12 light:dark (LD) cycle were given bilateral injections of the neurotoxin n-methyl-D-L-aspartic acid (NMA) or vehicle aimed at the vSPVZ; cells in the SCN are resistant to NMA, which kills neurons in other brain regions, but leaves fibers of passage intact. vSPVZ-damaged grass rats exhibited highly unstable patterns of activity in constant darkness (DD) and in the LD cycle that followed. However, crepuscular bouts of activity could be seen in all animals with vSPVZ lesions. Damage to the vSPVZ reduced cFos expression in this area but not in the SCN. Using correlational analyses, we found that the number of cFos-ir cells in the vSPVZ was unrelated to several parameters of the activity rhythms during the initial post-surgical period, when animals were in LD. However, the number of cells expressing cFos in the vSPVZ was positively correlated with general activity during the subjective day relative to the subjective night when the animals were switched to DD, and this pattern persisted when a LD cycle was reinstated. Also, the number of cFos-ir cells in the vSPVZ was negatively correlated with the strength of rhythmicity in DD and the number of days required to re-entrain to a LD cycle following several weeks in DD. These data suggest that the vSPVZ emits signals important for the expression of stable diurnal activity patterns in grass rats, and that species differences in these signals may contribute to differences in behavioral and physiological rhythms of diurnal and nocturnal mammals. (Author correspondence: mschw009@umaryland.edu ).

Daily rhythms and sex differences in vasoactive intestinal polypeptide, VIPR2 receptor and arginine vasopressin mRNA in the suprachiasmatic nucleus of a diurnal rodent, Arvicanthis niloticus.

Diurnal and nocturnal animals differ with respect to the time of day at which the ovulatory surge in luteinizing hormone occurs. In some species this is regulated by the suprachiasmatic nucleus (SCN), the primary circadian clock, via cells that contain vasoactive intestinal polypeptide (VIP) and vasopressin (AVP). Here, we evaluated the hypothesis that chronotype differences in the timing of the luteinizing hormone surge are associated with rhythms in expression of the genes that encode these neuropeptides. Diurnal grass rats (Arvicanthis niloticus) were housed in a 12/12-h light-dark cycle and killed at one of six times of day (Zeitgeber time 1, 5, 9, 13, 17, 21; ZT 0 = lights-on). In-situ hybridization was used to compare levels of vip, avp and VIP receptor mRNA (vipr2) in the SCN of intact females, ovariectomized females, ovariectomized females given estradiol and intact males. We found a sex difference in vip rhythms with a peak occurring at ZT 13 in males and ZT 5 in intact females. In all groups avp mRNA rhythms peaked during the day, from ZT 5 to ZT 9, and had a trough in the dark at ZT 21. There was a modest rhythm and sex difference in the pattern of vipr2. Most importantly, the patterns of each of these SCN rhythms relative to the light-dark cycle resembled those seen in nocturnal rodents. Chronotype differences in timing of neuroendocrine events associated with ovulation are thus likely to be generated downstream of the SCN.

Individual differences in rhythms of behavioral sleep and its neural substrates in Nile grass rats.

Laboratory populations of grass rats (Arvicanthis niloticus) housed with a running wheel show considerable variation in patterns of locomotor activity. At the extremes are "day-active" (DA) animals with a monophasic distribution of running throughout the light phase and "night-active" (NA) animals exhibiting a biphasic pattern with an extended peak at the beginning of the dark phase and a brief peak shortly before lights-on. Here, the authors use this intraspecific variation to explore interactions between circadian and homeostatic influences on sleep and the effects of these interactions on the activity of brain regions involved in sleep regulation. Male animals were singly housed with running wheels in a 12:12 LD cycle, videotaped for 24 h, and perfused at ZT 4 or 16. Behavioral sleep was scored from the videotapes, and brains were processed for cFos immunoreactivity (cFos-ir). Sleep duration within the light and dark phases was higher in NA and DA animals, respectively, but these groups did not differ with respect to total sleep. In both groups, sleep bouts were shortest in the light phase and longest between ZT 20 and ZT 23. In the ventrolateral preoptic area (VLPO), cFos-ir was higher at ZT 16 than at ZT 4 in DA but not NA grass rats, and it was correlated with behavioral sleep at ZT 16 but not ZT 4. In OXA neurons, cFos-ir was high at ZT 4 in DA grass rats and at ZT 16 in NA grass rats, and it was correlated with behavioral sleep at both times. In the lower subparaventricular zone (LSPV), cFos-ir was higher at ZT 16 in both DA and NA animals, and it was unrelated to behavioral sleep. Thus, patterns of cFos-ir in the LSPV and OXA neurons were most tightly linked to time and sleep, respectively, whereas cFos-ir in the VLPO was influenced by an interaction between these 2 variables.

Differences in the suprachiasmatic nucleus and lower subparaventricular zone of diurnal and nocturnal rodents.

Diurnal and nocturnal species are profoundly different in terms of the temporal organization of daily rhythms in physiology and behavior. The neural bases for these divergent patterns are at present unknown. Here we examine functional differences in the suprachiasmatic nucleus (SCN) and one of its primary targets in a diurnal rodent, the unstriped Nile grass rat (Arvicanthis niloticus) and in a nocturnal one, the laboratory rat (Rattus norvegicus). Grass rats and laboratory rats were housed in a 12:12 light:dark cycle, and killed at six time points. cFos-immunoreactive rhythms in the SCN of grass rats and laboratory rats were similar to those reported previously, with peaks early in the light phase and troughs in the dark phase. However, cFos-immunoreactivity in the lower subparaventricular zone (LSPV) of grass rats rose sharply 5 h into the dark phase, and remained high through the first hour after light onset, whereas in laboratory rats it peaked 1 h after light onset and was low at all other sampling times. Daily cFos rhythms in both the SCN and the LSPV persisted in grass rats, but not in laboratory rats, after extended periods in constant darkness. In grass rats, the endogenous cFos rhythm in the LSPV, but not the SCN, was present both in calbindin-positive and in calbindin-negative cells. Cells that expressed cFos at night in the region of the LSPV in grass rats were clearly outside of the boundaries of the SCN as delineated by Nissl stain and immunoreactivity for vasopressin and vasoactive intestinal peptide. The LSPV of the grass rat, a region that receives substantial input from the SCN, displays a daily rhythm in cFos expression that differs from that of laboratory rats with respect to its rising phase, the duration of the peak and its dependence on a light/dark cycle. These characteristics may reflect the existence of mechanisms in the LSPV that enable it to modulate efferent SCN signals differently in diurnal and nocturnal species.

Individual differences in wheel-running rhythms are related to temporal and spatial patterns of activation of orexin A and B cells in a diurnal rodent (Arvicanthis niloticus).

This study investigated the relationship between the orexins and patterns of activity in the diurnal Nile grass rat, Arvicanthis niloticus. Some individuals of this species switch to a more nocturnal pattern when given access to a running wheel, while others continue to be most active during the day. In both day- and night-active grass rats, the percentages of orexin A (OXA) and orexin B (OXB) cells expressing Fos were highest when animals were actively running in wheels. In night-active animals, removal of the running wheel significantly decreased OXA and OXB cell Fos expression. Additionally, in night-active animals, clear regional differences were apparent. In these animals the presence of a wheel induced higher percentages of Fos in both OXA and OXB cells in medial regions of the lateral hypothalamus than in lateral regions. In night-active animals without access to wheels, this medial-lateral gradient was present only in OXA cells. No regional differences were observed in day-active animals. This study demonstrates that individual differences in the patterns of activation of OXA and OXB cell populations are related to differences in the temporal pattern of wheel running. We also present evidence that orexin cells have projections to the intergeniculate leaflet that appear to make contact with neuropeptide-Y cells. We discuss the possibility that these fibers may be involved in relaying feedback regarding the activity state of the animal to the circadian system through these projections.

Fos rhythms in the hypothalamus of Rattus and Arvicanthis that exhibit nocturnal and diurnal patterns of rhythmicity.

This study compared patterns of Fos expression within the suprachiasmatic nucleus (SCN), the region immediately dorsal to the SCN (the lower subparaventricular zone, LSPV), and the supraoptic nucleus (SON) of grass rats (Arvicanthis niloticus) and lab rats (Rattus norvegicus). Among grass rats we also compared individuals exhibiting nocturnal and diurnal patterns of wheel running. In the SCN of both groups of grass rats, as well as laboratory rats, Fos was elevated during the light compared to the dark portions of the day, and was expressed in 7-12% of cells containing vasoactive intestinal polypeptide (VIP). Fos was higher in the LSPV during the night compared to the day in both forms of grass rats but not in laboratory rats. In the SON, Fos rose from day to night in the diurnal grass rats and in laboratory rats, but not in nocturnal grass rats. These patterns are consistent with the hypothesis that VIP cells in the SCN function similarly in nocturnal and diurnal rodents, but that the SON and the region dorsal to the SCN are associated with intra and interspecific differences in rhythmicity, respectively.

Phase response curve and light-induced fos expression in the suprachiasmatic nucleus and adjacent hypothalamus of Arvicanthis niloticus.

This article describes the phase response curve (PRC), the effect of light on Fos immunoreactivity (Fos-IR) in the suprachiasmatic nucleus (SCN), and the effect of SCN lesions on circadian rhythms in the murid rodent, Arvicanthis niloticus. In this species, all individuals are diurnal when housed without a running wheel, but running in a wheel induces a nocturnal pattern in some individuals. First, the authors characterized the PRC in animals with either the nocturnal or diurnal pattern. Both groups of animals were less affected by light during the middle of the subjective day than during the night and were phase delayed and phase advanced by pulses in the early and late subjective night, respectively. Second, the authors characterized the Fos response to light at circadian times 5, 14, or 22. Light induced an increase in Fos-IR within the SCN during the subjective night but not subjective day; this effect was especially pronounced in the ventral SCN, where retinal inputs are most concentrated, but was also evident in other regions. Both light and time influenced Fos-IR within the lower subparaventricular area. Third, SCN lesions caused animals to become arrhythmic when housed in a light-dark cycle as well as constant darkness. In summary, Arvicanthis appear to be very similar to nocturnal rodents with respect to their PRC, temporal patterns of light-induced Fos expression in the SCN, and the effects of SCN lesions on activity rhythms.

Patterns of wheel running are related to Fos expression in neuropeptide-Y-containing neurons in the intergeniculate leaflet of Arvicanthis niloticus.

A variety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGL of Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.

Calbindin and Fos within the suprachiasmatic nucleus and the adjacent hypothalamus of Arvicanthis niloticus and Rattus norvegicus.

The suprachiasmatic nucleus is the site of the primary circadian pacemaker in mammals. The lower sub paraventricular zone that is dorsal to and receives input from the suprachiasmatic nucleus may also play a role in the regulation of circadian rhythms. Calbindin has been described in the suprachiasmatic nucleus of some mammals, and may be important in the control of endogenous rhythms. In the first study we characterized calbindin-expressing cells in the suprachiasmatic nucleus and lower sub-paraventricular zone of nocturnal and diurnal rodents. Specifically, Rattus norvegicus was compared to Arvicanthis niloticus, a primarily diurnal species within which some individuals exhibit nocturnal patterns of wheel running. Calbindin-immunoreactive cells were present in the suprachiasmatic nucleus of Arvicanthis and were most concentrated within its central region but were relatively sparse in the suprachiasmatic nucleus of Rattus. Calbindin-expressing cells were present in the lower sub-paraventricular zone of both species. In the second study we evaluated Fos expression within calbindin-immunoreactive cells in nocturnal Rattus and in Arvicanthis that were either diurnal or nocturnal with respect to wheel-running. All animals were kept on a 12:12 light/dark cycle and perfused at either 4h after lights-on or 4h after lights-off. In the suprachiasmatic nucleus in both species, Fos expression was elevated during the day relative to the night but less than 1% of calbindin cells contained Fos in Arvicanthis, compared with 13-17% in Rattus. In the lower sub-paraventricular zone of both species, 9-14% of calbindin cells expressed Fos, and this proportion did not change as a function of time. Among Arvicanthis, the number of calbindin expressing neurons in the lower sub-paraventricular zone was influenced by an interaction between the wheel running patterns (nocturnal vs diurnal) and time of day. Thus, the number of calbindin-positive cells within the suprachiasmatic nucleus differed in Arvicanthis and Rattus, whereas the number of calbindin-positive cells within the lower sub-paraventricular zone differed in nocturnal and diurnal Arvicanthis. Our examination of R. norvegicus and A. niloticus suggests potentially important relationships between calbindin-containing neurons and whether animals are nocturnal or diurnal. Specifically, rats had more Fos expression in calbindin containing cells in the suprachiasmatic nucleus than Arvicanthis. In contrast, Arvicanthis exhibiting diurnal and nocturnal patterns of wheel-running differed in the number of calbindin-containing cells in the lower sub-paraventricular zone, dorsal to the suprachiasmatic nucleus.

Rhythms in Fos expression in brain areas related to the sleep-wake cycle in the diurnal Arvicanthis niloticus.

Most mammals show daily rhythms in sleep and wakefulness controlled by the primary circadian pacemaker, the suprachiasmatic nucleus (SCN). Regardless of whether a species is diurnal or nocturnal, neural activity in the SCN and expression of the immediate-early gene product Fos increases during the light phase of the cycle. This study investigated daily patterns of Fos expression in brain areas outside the SCN in the diurnal rodent Arvicanthis niloticus. We specifically focused on regions related to sleep and arousal in animals kept on a 12:12-h light-dark cycle and killed at 1 and 5 h after both lights-on and lights-off. The ventrolateral preoptic area (VLPO), which contained cells immunopositive for galanin, showed a rhythm in Fos expression with a peak at zeitgeber time (ZT) 17 (with lights-on at ZT 0). Fos expression in the paraventricular thalamic nucleus (PVT) increased during the morning (ZT 1) but not the evening activity peak of these animals. No rhythm in Fos expression was found in the centromedial thalamic nucleus (CMT), but Fos expression in the CMT and PVT was positively correlated. A rhythm in Fos expression in the ventral tuberomammillary nucleus (VTM) was 180 degrees out of phase with the rhythm in the VLPO. Furthermore, Fos production in histamine-immunoreactive neurons of the VTM cells increased at the light-dark transitions when A. niloticus show peaks of activity. The difference in the timing of the sleep-wake cycle in diurnal and nocturnal mammals may be due to changes in the daily pattern of activity in brain regions important in sleep and wakefulness such as the VLPO and the VTM.

A morning surge in plasma luteinizing hormone coincides with elevated Fos expression in gonadotropin-releasing hormone-immunoreactive neurons in the diurnal rodent, Arvicanthis niloticus.

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

Daily rhythms of Fos expression in hypothalamic targets of the suprachiasmatic nucleus in diurnal and nocturnal rodents.

Little is known about the differences in the neural substrates of circadian rhythms that are responsible for the maintenance of differences between diurnal and nocturnal patterns of activity in mammals. In both groups of animals, the suprachiasmatic nucleus (SCN) functions as the principal circadian pacemaker, and surprisingly, several correlates of neuronal activity in the SCN show similar daily patterns in diurnal and nocturnal species. In this study, immunocytochemistry was used to monitor daily fluctuations in the expression of the nuclear phosphoprotein Fos in the SCN and in hypothalamic targets of the SCN axonal outputs in the nocturnal laboratory rat and in the diurnal murid rodent, Arvicanthis niloticus. The daily patterns of Fos expression in the SCN were very similar across the two species. However, clear species differences were seen in regions of the hypothalamus that receive inputs from the SCN including the subparaventricular zone. These results indicate that differences in the circadian system found downstream from the SCN contribute to the emergence of a diurnal or nocturnal profile in mammals.

Suprachiasmatic nucleus and intergeniculate leaflet in the diurnal rodent Octodon degus: retinal projections and immunocytochemical characterization.

The neural connections and neurotransmitter content of the suprachiasmatic nucleus and intergeniculate leaflet have been characterized thoroughly in only a few mammalian species, primarily nocturnal rodents. Few data are available about the neural circadian timing system in diurnal mammals, particularly those for which the formal characteristics of circadian rhythms have been investigated. This paper describes the circadian timing system in the diurnal rodent Octodon degus, a species that manifests robust circadian responses to photic and non-photic (social) zeitgebers. Specifically, this report details: (i) the distribution of six neurotransmitters commonly found in the suprachiasmatic nucleus and intergeniculate leaflet; (ii) the retinohypothalamic tract; (iii) the geniculohypothalamic tract; and (iv) retinogeniculate projections in O. degus. Using immunocytochemistry, neuropeptide Y-immunoreactive, serotonin-immunoreactive and [Met]enkephalin-immunoreactive fibers and terminals were detected in and around the suprachiasmatic nucleus; vasopressin-immunoreactive cell bodies were found in the dorsomedial and ventral suprachiasmatic nucleus; vasoactive intestinal polypeptide-immunoreactive cell bodies were located in the ventral suprachiasmatic nucleus; [Met]enkephalin-immunoreactive cells were located sparsely throughout the suprachiasmatic nucleus; and substance P-immunoreactive fibers and terminals were detected in the rostral suprachiasmatic nucleus and surrounding the nucleus throughout its rostrocaudal dimension. Neuropeptide Y-immunoreactive and [Met]enkephalin-immunoreactive cells were identified in the intergeniculate leaflet and ventral lateral geniculate nucleus, as were neuropeptide Y-immunoreactive, [Met]enkephalin-immunoreactive, serotonin-immunoreactive and substance P-immunoreactive fibers and terminals. The retinohypothalamic tract innervated both suprachiasmatic nuclei equally; in contrast, retinal innervation to the lateral geniculate nucleus, including the intergeniculate leaflet, was almost exclusively contralateral. Bilateral electrolytic lesions that destroyed the intergeniculate leaflet depleted the suprachiasmatic nucleus of virtually all neuropeptide Y- and [Met]enkephalin-stained fibers and terminals, whereas unilateral lesions reduced fiber and terminal staining by approximately half. Thus, [Met]enkephalin-immunoreactive and neuropeptide Y-immunoreactive cells project equally and bilaterally from the intergeniculate leaflet to the suprachiasmatic nucleus via the geniculohypothalamic tract in degus. This is the first report examining the neural circadian system in a diurnal rodent for which formal circadian properties have been described. The data indicate that the neural organization of the circadian timing system in degus resembles that of the most commonly studied nocturnal rodents, golden hamsters and rats. Armed with such data, one can ascertain differences in the functional organization of the circadian system between diurnal and nocturnal mammals.

Fos expression in the sleep-active cell group of the ventrolateral preoptic area in the diurnal murid rodent, Arvicanthis niloticus.

The ventrolateral preoptic area (VLPO) of the nocturnal laboratory rat receives direct input from the retina and is active during sleep; however, nothing is known about VLPO function in day-active (diurnal) species. In the first study, we used 24-h videotaping of Arvicanthis niloticus, a diurnal murid rodent, to estimate the distribution of sleep and wakefulness across a 12:12 light-dark cycle. Based on behavioral data, A. niloticus were perfused at a time when the animals are inactive (zeitgeber time (ZT) 20) or at a time when they are awake and active (ZT 23). The brains were processed for immunocytochemistry for Fos, an immediate early gene product used as an index of neural activity. Animals had more Fos-immunoreactive (Fos+) cells in the VLPO at ZT 20 than at ZT 23. The pattern of change in Fos expression seen in this area suggest that the VLPO serves the same function in A. niloticus as in rats. Eye injections of cholera toxin (beta subunit) were used to identify the retinal inputs to the VLPO of A. niloticus. In these animals, the VLPO had only very sparse retinal inputs compared to the rat. Together, these results raise the possibility that inputs from the suprachiasmatic nucleus (SCN) or the retina affect neuronal activity in the VLPO differently in rats and A. niloticus, thereby, contributing to differences in their sleep/wake patterns.

Fos expression within vasopressin-containing neurons in the suprachiasmatic nucleus of diurnal rodents compared to nocturnal rodents.

The underlying neural causes of the differences between nocturnal and diurnal animals with respect to their patterns of rhythmicity have not yet been identified. These differences could be due to differences in some subpopulation of neurons within the suprachiasmatic nucleus (SCN) or to differences in responsiveness to signals emanating from the SCN. The experiments described in this article were designed to address the former hypothesis by examining Fos expression within vasopressin (VP) neurons in the SCN of nocturnal and diurnal rodents. Earlier work has shown that within the SCN of the diurnal rodent Arvicanthis niloticus, approximately 30% of VP-immunoreactive (IR) neurons express Fos during the day, whereas Fos rarely is expressed in VP-IR neurons in the SCN of nocturnal rats. However, in earlier studies, rats were housed in constant darkness and pulsed with light, whereas Arvicanthis were housed in a light:dark (LD) cycle. To provide data from rats that would permit comparisons with A. niloticus, the first experiment examined VP/Fos double labeling in the SCN of rats housed in a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase. Fos was significantly elevated in the SCN of animals sacrificed during the light compared to the dark phase, but virtually no Fos at either time was found in VP-IR neurons, confirming that the SCN of rats and diurnal Arvicanthis are significantly different in this regard. The authors also evaluated the relationship between this aspect of SCN function and diurnality by examining Fos-IR and VP-IR in diurnal and nocturnal forms of Arvicanthis. In this species, most individuals exhibit diurnal wheel-running rhythms, but some exhibit a distinctly different and relatively nocturnal pattern. The authors have bred their laboratory colony for this trait and used animals with both patterns in this experiment. They examined Fos expression within VP-IR neurons in the SCN of both nocturnal and diurnal A. niloticus kept on a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase, and brains were processed for immunohistochemical identification of Fos and VP. Both the total number of Fos-IR cells and the proportion of VP-IR neurons containing Fos (20%) were higher during the day than during the night. Neither of these parameters differed between nocturnal and diurnal animals. The implications of these findings are discussed.

The suprachiasmatic nucleus and intergeniculate leaflet of Arvicanthis niloticus, a diurnal murid rodent from East Africa.

Little is known about the neural substrates controlling circadian rhythms in day-active compared to night-active mammals primarily because of the lack of a suitable diurnal rodent with which to address the issue. The murid rodent, Arvicanthis niloticus, was recently shown to exhibit a predominantly diurnal pattern of activity and body temperature, and may be suitable for research on the neural mechanisms underlying circadian rhythms. This paper describes, in A. niloticus, the anatomy of two neural structures that play important roles in the control of circadian rhythms, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL). Immunohistochemical techniques were used to examine the distribution of neuroactive peptides in the SCN and IGL, and retinal projections to these structures were traced with anterograde transport of the beta subunit of cholera toxin. In A. niloticus, distinct subdivisions of the SCN contained cell bodies with immunoreactive (IR) vasopressin, vasoactive intestinal polypeptide, gastrin-releasing peptide, and corticotropin-releasing factor. The SCN did not contain cell bodies with met-enkephalin-IR and substance P-IR, but did contain fibers with substance P-IR and neuropeptide Y-IR. Retinal fibers were present throughout the SCN, but were most densely concentrated along its ventral edge, particularly in the contralateral SCN. Retinal fibers also extended to a variety of hypothalamic regions outside the SCN, including the supraoptic nucleus and the subparaventricular region. The IGL contained cells with neuropeptide Y-IR and enkephalin-IR cells. Retinal fibers projected to both the ipsilateral and contralateral IGL. The anatomy of the SCN and IGL were compared and contrasted with that previously described for other nocturnal and diurnal species.

The expression of Fos within the suprachiasmatic nucleus of the diurnal rodent Arvicanthis niloticus.

Rhythms in the expression of the nuclear phosphoprotein Fos, have been demonstrated in the suprachiasmatic nucleus (SCN) of nocturnal rodents. When rats are housed in a 12:12-h light/dark (LD) cycle the number of Fos-immunoreactive (-IR) cells within the SCN is higher during the day than at night [9,23]. In the two experiments reported here, Fos-IR was examined in the SCN of a diurnal murid rodent, Arvicanthis niloticus. First, thirty-six adult male A. niloticus housed in a 12:12-h LD cycle were perfused at six equally spaced time points beginning 1 h after lights on (n=6 per time point). Brains were sectioned and treated with immunohistochemical procedures for the identification of Fos. The number of Fos-IR cells in the SCN varied significantly as a function of time, and was highest 1 h after lights on and decreased thereafter. The distribution of Fos-IR within the SCN overlapped with that of arginine-vasopressin-IR (AVP-IR) and vasoactive intestinal peptide-IR (VIP-IR), but not with that of gastrin-releasing peptide-IR (GRP-IR). In the second study, double-labeling techniques revealed extensive Fos expression within SCN neurons containing AVP-IR, but not neurons containing GRP-IR. In conclusion, although the overall rhythm of Fos-IR in the SCN is similar in diurnal and nocturnal rodents, differences may exist with respect to the relative distribution of Fos-immunoreacte cells within different SCN cell populations.