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.     

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.     

Rhythms in a diurnal brain

The neural mechanisms governing circadian rhythms generate patterns of behavior and physiology that are very different in diurnal and nocturnal species. Here we review data bearing on the issue of where and how in the brain these differences might be generated. Molecular data from several species now confirm that the central circadian clock, located in the suprachiasmatic nucleus (SCN), is coupled to the light - dark cycle in the same manner in nocturnal and diurnal species, indicating that the fundamental differences arise from mechanisms coupling the clock to effector systems. Major differences in this coupling become apparent only when one steps beyond the SCN to look at brain regions that directly or indirectly receive input from it. This review focuses on our work on brain regions and cell populations to which the SCN projects in the diurnal species Arvicanthis niloticus (Nile grass rats). We have found rhythms in the numbers of cells containing cFos, or PER1, in a number of these regions, and the patterns of these rhythms are always different from those seen in nocturnal laboratory rats. In some areas these rhythms are simply inverted in the two species, but in other extra-SCN regions the phase of the rhythms in these two species differs in less extreme ways. Taken together, these data suggest that there is no single simple switch that causes some animals to be nocturnal and others to be diurnal. Rather, the differences likely emerge through a variety of mechanisms operating within and downstream of the cells to which the SCN projects.     

Nocturnal and diurnal rhythms in the unstriped Nile rat, Arvicanthis niloticus.

In a laboratory population of unstriped Nile grass rats, Arvicanthis niloticus, individuals with two distinctly different patterns of wheel-running exist. One is diurnal and the other is relatively nocturnal. In the first experiment, the authors found that these patterns are strongly influenced by parentage and by sex. Specifically, offspring of two nocturnal parents were significantly more likely to express a nocturnal pattern of wheel-running than were offspring of diurnal parents, and more females than males were nocturnal. In the second experiment, the authors found that diurnal and nocturnal wheel-runners were indistinguishable with respect to the timing of postpartum mating, which always occurred in the hours before lights-on. Here they also found that both juvenile and adult A. niloticus exhibited diurnal patterns of general activity when housed without a wheel, even if they exhibited nocturnal activity when housed with a wheel. In the third experiment, the authors discovered that adult female A. niloticus with nocturnal patterns of wheel-running were also nocturnal with respect to general activity and core body temperature when a running wheel was available, but they were diurnal when the running wheel was removed. Finally, a field study revealed that all A. niloticus were almost exclusively diurnal in their natural habitat. Together these results suggest that individuals of this species are fundamentally diurnal but that access to a running wheel shifts some individuals to a nocturnal pattern.     

A daily rhythm in mating behavior in a diurnal murid rodent Arvicanthis niloticus

The time of day at which mating occurs is dramatically different in diurnal compared to nocturnal rodents. We used a diurnal murid rodent, Arvicanthis niloticus, to determine if inverted rhythms in responsiveness to hormones contribute to this difference. Male and hormone-primed female grass rats were tested for mating behavior at four different times of day (ZT 5, 11, 17, 23; ZT 0=lights-on). In females, there was considerable inter-individual variability with respect to patterns of responsiveness to hormones. Overall, the lordosis quotient (LQ) was rhythmic with a single peak just before lights-on (ZT 23); however, while roughly half of the females (7/15) exhibited this clear daily rhythm, the remaining animals (8/15) had relatively high LQs that did not change as a function of time. Males had their shortest ejaculation latencies and their highest number of ejaculations at ZT 23. Rhythms in mount frequency and post-ejaculatory refractory period were bimodal, with peaks around lights-on and -off (ZT 23 and 11). This temporal pattern of mounting behavior closely parallels previously documented patterns of general activity, whereas rhythms in the more reflexive components of sex behavior (LQ and ejaculation) had more restricted peaks that coincided with just the onset of rhythms in general activity. These rhythms in sexual behavior are essentially reversed relative to those previously documented in lab rats.     

Nocturnal activity in a diurnal rodent (Arvicanthis niloticus): the importance of masking

It is known that day-active Nile grass rats, Arvicanthis niloticus, increase the amount of activity in the night relative to that in the day when provided with running wheels. This was confirmed in the present study. Animals without a wheel displayed 69.0% of their general activity in the L phase of a 12:12 h light-dark cycle; animals provided with wheels had only 48.6% of their wheel revolutions in the light. The contribution of direct (masking) responses to light to the increased nocturnality of animals with wheels was examined in two experiments. In experiment 1, masking was tested by exposing the animals to repeated cycles of 30 min of entraining light and 30 min of a different, usually dimmer light, during the L phase of a 12:12 h light-dark cycle. For animals with wheels, there was more running during the 30-min pulses of dim light or darkness than during the 30-min periods of entraining light. In contrast, for animals without wheels, there was more general activity during the 30-min periods of entraining light than during the 30-min pulses of dim light or darkness. In experiment 2, the animals were first exposed to a 12:12 h light-dark cycle and then put on a 1:10:1:12 h LDLD skeleton photoperiod. Animals with wheels increased their running during the subjective day of the skeleton photoperiod compared to that in the actual day of the 12:12 h light-dark cycle. Animals without wheels showed similar levels of general activity during the subjective day of the skeleton photoperiod and the actual day of the 12:12 h cycle. These experiments demonstrate that when Nile rats have running wheels, their increased nocturnal activity is associated with an increased suppression of locomotion in direct response to light. It is possible that changes in masking responses to light may be an essential and integral component of switching between diurnal and nocturnal activity profiles.     

Functional and anatomical variations in retinorecipient brain areas in Arvicanthis niloticus and Rattus norvegicus: implications for the circadian and masking systems

ABSTRACT

Daily rhythms in light exposure influence the expression of behavior by entraining circadian rhythms and through its acute effects on behavior (i.e., masking). Importantly, these effects of light are dependent on the temporal niche of the organism; for diurnal organisms, light increases activity, whereas for nocturnal organisms, the opposite is true. Here we examined the functional and morphological differences between diurnal and nocturnal rodents in retinorecipient brain regions using Nile grass rats (Arvicanthis niloticus) and Sprague-Dawley (SD) rats (Rattus norvegicus), respectively. We established the presence of circadian rhythmicity in cFOS activation in retinorecipient brain regions in nocturnal and diurnal rodents housed in constant dark conditions to highlight different patterns between the temporal niches. We then assessed masking effects by comparing cFOS activation in constant darkness (DD) to that in a 12:12 light/dark (LD) cycle, confirming light responsiveness of these regions during times when masking occurs in nature. The intergeniculate leaflet (IGL) and olivary pretectal nucleus (OPN) exhibited significant variation among time points in DD of both species, but their expression profiles were not identical, as SD rats had very low expression levels for most timepoints. Light presentation in LD conditions induced clear rhythms in the IGL of SD rats but eliminated them in grass rats. Additionally, grass rats were the only species to demonstrate daily rhythms in LD for the habenula and showed a strong response to light in the superior colliculus. Structurally, we also analyzed the volumes of the visual brain regions using anatomical MRI, and we observed a significant increase in the relative size of several visual regions within diurnal grass rats, including the lateral geniculate nucleus, superior colliculus, and optic tract. Altogether, our results suggest that diurnal grass rats devote greater proportions of brain volume to visual regions than nocturnal rodents, and cFOS activation in these brain regions is dependent on temporal niche and lighting conditions.     

Ghrelin agonists impact on Fos protein expression in brain areas related to food intake regulation in male C57BL/6 mice

Many peripheral substances, including ghrelin, induce neuronal activation in the brain. In the present study, we compared the effect of subcutaneously administered ghrelin and its three stable agonists: Dpr³ghr ([Dpr(N-octanoyl)³] ghrelin) (Dpr - diaminopropionic acid), YA GHRP-6 (H-Tyr-Ala-His-DTrp-Ala-Trp-DPhe-Lys-NH₂), and JMV1843 (H-Aib-DTrp-D-gTrp-CHO) on the Fos expression in food intake-responsive brain areas such as the hypothalamic paraventricular (PVN) and arcuate (ARC) nuclei, the nucleus of the solitary tract (NTS), and area postrema (AP) in male C57BL/6 mice. Immunohistochemical analysis showed that acute subcutaneous dose of each substance (5 mg/kg b.w.), which induced a significant food intake increase, elevated Fos protein expression in all brain areas studied. Likewise ghrelin, each agonist tested induced distinct Fos expression overall the PVN. In the ARC, ghrelin and its agonists specifically activated similarly distributed neurons. Fos occurrence extended from the anterior (aARC) to middle (mARC) ARC region. In the latter part of the ARC, the Fos profiles were localized bilaterally, especially in the ventromedial portions of the nucleus. In the NTS, all substances tested also significantly increased the number of Fos profiles in neurons, which also revealed specific location, i.e., in the NTS dorsomedial subnucleus (dmNTS) and the area subpostrema (AsP). In addition, cells located nearby the NTS, in the AP, also revealed a significant increase in number of Fos-activated cells. These results demonstrate for the first time that ghrelin agonists, regardless of their different chemical nature, have a significant and similar activating impact on specific groups of neurons that can be a part of the circuits involved in the food intake regulation. Therefore there is a real potency for ghrelin agonists to treat cachexia and food intake disorders. Thus, likewise JMV1843, the other ghrelin agonists represent substances that might be involved in trials for clinical purposes.     

Effect of tranquilizers on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of Arvicanthis niloticus

The effect of reserpine and meprobamate on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of the kusu rat, Arvicanthis niloticus, was studied. The total acetylcholine content and acetylcholinesterase activity were determined 1 hr after i.p. injection of different doses of reserpine (0.25, 0.5 and 1 mg/ml/100 g body wt) and meprobamate (6.25, 12.5 and 25 mg/ml/100 g body wt). The effect of different time intervals (1, 10, 30 min, 1, 2.5, 5, 8, 12, 24 and 48 hr) on the total acetylcholine content and acetylcholinesterase activity was investigated after i.p. injection of 0.5 mg of reserpine and 12.5 mg of meprobamate/ml/100 g body wt. Both reserpine and meprobamate caused an increase in the total ACh content in the brain tissue of Arvicanthis niloticus which was suggested to be due to a decrease in the release of ACh, since both reserpine and meprobamate inhibited AChE activity after some tested periods. The effect of meprobamate was observed to be stronger than that of reserpine.     

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.     

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.     

Chloramphenicol decreases brain glucose utilization and modifies the sleep-wake cycle architecture in rats

We studied the effects of chloramphenicol on brain glucose utilization and sleep-wake cycles in rat. After slightly anaesthetized animals were injected with [18F]fluoro-2-deoxy-D-glucose, we acquired time-concentration curves from three radiosensitive beta microprobes inserted into the right and left frontal cortices and the cerebellum, and applied a three-compartment model to calculate the cerebral metabolic rates for glucose. The sleep-wake cycle architecture was analysed in anaesthetic-free rats by recording electroencephalographic and electromyographic signals. Although chloramphenicol is a well-established inhibitor of oxidative phosphorylation, no compensatory increase in glucose utilization was detected in frontal cortex. Instead, chloramphenicol induced a significant 23% decrease in the regional cerebral metabolic rate for glucose. Such a metabolic response indicates a potential mismatch between energy supply and neuronal activity induced by chloramphenicol administration. Regarding sleep-wake states, chloramphenicol treatment was followed by a 64% increase in waking, a 20% decrease in slow-wave sleep, and a marked 59% loss in paradoxical sleep. Spectral analysis of the electroencephalogram indicates that chloramphenicol induces long-lasting modifications of delta-band power during slow-wave sleep.     

Acute Behavioral Responses to Light and Darkness in Nocturnal Mus musculus and Diurnal Arvicanthis niloticus

The term masking refers to immediate responses to stimuli that override the influence of the circadian timekeeping system on behavior and physiology. Masking by light and darkness plays an important role in shaping an organism's daily pattern of activity. Nocturnal animals generally become more active in response to darkness (positive masking) and less active in response to light (negative masking), and diurnal animals generally have opposite patterns of response. These responses can vary as a function of light intensity as well as time of day. Few studies have directly compared masking in diurnal and nocturnal species, and none have compared rhythms in masking behavior of diurnal and nocturnal species. Here, we assessed masking in nocturnal mice (Mus musculus) and diurnal grass rats (Arvicanthis niloticus). In the first experiment, animals were housed in a 12:12 light-dark (LD) cycle, with dark or light pulses presented at 6 Zeitgeber times (ZTs; with ZT0 = lights on). Light pulses during the dark phase produced negative masking in nocturnal mice but only at ZT14, whereas light pulses resulted in positive masking in diurnal grass rats across the dark phase. In both species, dark pulses had no effect on behavior. In the 2nd experiment, animals were kept in constant darkness or constant light and were presented with light or dark pulses, respectively, at 6 circadian times (CTs). CT0 corresponded to ZT0 of the preceding LD cycle. Rhythms in masking responses to light differed between species; responses were evident at all CTs in grass rats but only at CT14 in mice. Responses to darkness were observed only in mice, in which there was a significant increase in activity at CT 22. In the 3rd experiment, animals were kept on a 3.5:3.5-h LD cycle. Surprisingly, masking was evident only in grass rats. In mice, levels of activity during the light and dark phases of the 7-h cycle did not differ, even though the same animals had responded to discrete photic stimuli in the first 2 experiments. The results of the 3 experiments are discussed in terms of their methodological implications and for the insight they offer into the mechanisms and evolution of diurnality.     

Effect of thiopental sodium and barbitone sodium on the total acetylcholine content and acetylcholinesterase activity in the brain tissue of Arvicanthis niloticus

The total ACh content and AChE activity were determined 1 hr after the i.p. injection of different doses of thiopental sodium (5, 10 and 20 mg/ml/100 g body wt) and barbitone sodium (20, 40 and 80 mg/ml/100 g body wt). The effect of different time intervals (1 min, 10 min, 30 min, 1 hr, 2.5 hr, 5 hr, 8 hr, 12 hr, 24 hr and 48 hr) on the total ACh content and AChE activity was investigated after i.p. injection of 10 mg thiopental sodium and 40 mg barbitone sodium/ml/100 g body wt. Both thiopental sodium and barbitone sodium increased the total ACh content in the brain tissue of Arvicanthis niloticus. Both drugs inhibited the brain AChE activity. It is thought that the increase in the total ACh content in the brain tissue of Arvicanthis niloticus may be due to a decrease in the release of ACh from the neuronal tissue and a decrease in AChE activity.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Circadian regulation of gonadotropin-releasing hormone neurons and the preovulatory surge in luteinizing hormone in the diurnal rodent, Arvicanthis niloticus, and in a nocturnal rodent, Rattus norvegicus

Daily rhythms in the timing of the preovulatory surge and the display of reproductive behavior are reversed in diurnal and nocturnal rodents, but little is known about the neural mechanisms underlying these differences. We examined this issue by comparing a diurnal murid rodent, Arvicanthis niloticus (the grass rat), to a nocturnal one, Rattus norvegicus (the lab rat). In the first study, we established that sequential estradiol and progesterone treatment induces a proestrous-like rise in LH secretion and in the percentage of GnRH neurons that express Fos in grass rats, as is the case in lab rats. Next, we tested the hypothesis that differences in the timing of estrus-related events in diurnal and nocturnal species are caused by differences in rhythms in responsiveness to steroid hormones. We found rhythms in GnRH neuron activity, as indicated by Fos, that were 12 hours out of phase in grass rats and lab rats. These patterns persisted in both species when animals were housed in constant darkness for 5 days, suggesting that they are driven by an endogenous circadian mechanism. These results indicate that steroid-primed grass rats and lab rats are similar with respect to the temporal relationship among estrus-related events, but that the timing of these events relative to the light-dark cycle is dramatically different and that this difference is caused by endogenous circadian mechanisms.