A comparative analysis of the distribution of immunoreactive orexin A and B in the brains of nocturnal and diurnal rodents

<p>Abstract</p> <p>Background</p> <p>The orexins (hypocretins) are a family of peptides found primarily in neurons in the lateral hypothalamus. Although the orexinergic system is generally thought to be the same across species, the orexins are involved in behaviors which show considerable interspecific variability. There are few direct cross-species comparisons of the distributions of cells and fibers containing these peptides. Here, we addressed the possibility that there might be important species differences by systematically examining and directly comparing the distribution of orexinergic neurons and fibers within the forebrains of species with very different patterns of sleep-wake behavior.</p> <p>Methods</p> <p>We compared the distribution of orexin-immunoreactive cell bodies and fibers in two nocturnal species (the lab rat, <it>Rattus norvegicus </it>and the golden hamster, <it>Mesocricetus auratus</it>) and two diurnal species (the Nile grass rat, <it>Arvicanthis niloticus </it>and the degu, <it>Octodon degus</it>). For each species, tissue from the olfactory bulbs through the brainstem was processed for immunoreactivity for orexin A and orexin B (hypocretin-1 and -2). The distribution of orexin-positive cells was noted for each species. Orexin fiber distribution and density was recorded and analyzed using a principal components factor analysis to aid in evaluating potential species differences.</p> <p>Results</p> <p>Orexin-positive cells were observed in the lateral hypothalamic area of each species, though there were differences with respect to distribution within this region. In addition, cells positive for orexin A but not orexin B were observed in the paraventricular nucleus of the lab rat and grass rat, and in the supraoptic nucleus of the lab rat, grass rat and hamster. Although the overall distributions of orexin A and B fibers were similar in the four species, some striking differences were noted, especially in the lateral mammillary nucleus, ventromedial hypothalamic nucleus and flocculus.</p> <p>Conclusion</p> <p>The orexin cell and fiber distributions observed in this study were largely consistent with those described in previous studies. However, the present study shows significant species differences in the distribution of orexin cell bodies and in the density of orexin-IR fibers in some regions. Finally, we note previously undescribed populations of orexin-positive neurons outside the lateral hypothalamus in three of the four species examined.</p

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical.This study was supported by the National Science Foundation grant (IOS-1051919) to LS and the Danish Biotechnology Center for Cellular Communication (JH)

Intergeniculate Leaflet Lesions Result in Differential Activation of Brain Regions Following the Presentation of Photic Stimuli in Nile Grass Rats

The intergeniculate leaflet (IGL) plays an important role in the entrainment of circadian rhythms and the mediation of acute behavioral responses to light (i.e., masking). Recently, we reported that IGL lesions in diurnal grass rats result in a reversal in masking responses to light as compared to controls. Here, we used Fos as a marker of neural activation to examine the mechanisms by which the IGL may influence this masking effect of light in grass rats. Specifically, we examined the patterns of Fos activation in retinorecipient areas and in brain regions that receive IGL inputs following 1-h light pulses given during the early night in IGL-lesioned and sham-operated grass rats. Three patterns emerged: (1) IGL lesions had no effect on the Fos response to light, (2) IGL lesions resulted in a reversal in Fos responses to light, and (3) IGL lesions resulted in a lack of a Fos response to light. Of specific interest were the suprachiasmatic nucleus (SCN) and the olivary pretectal nucleus (OPT), both of which are retinorecipient and connect reciprocally with the IGL. Light-induced Fos expression in the SCN was unaffected by IGL lesions, whereas the OPT exhibited a significant reduction in Fos expression following a light pulse in animals with IGL lesions. Altogether, our results suggest that the OPT, but not the SCN, exhibits a reversal in Fos responses to light following IGL lesions that reverse masking responses in diurnal grass rats. Our results suggest that interconnections between the IGL and downstream brain areas (e.g., OPT) may play a role in determining the direction of the behavioral response to light

Lesions of the Intergeniculate Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in Masking Responses to Photic Stimuli in the Nile Grass Rat

Light influences the daily patterning of behavior by entraining circadian rhythms and through its acute effects on activity levels (masking). Mechanisms of entrainment are quite similar across species, but masking can be very different. Specifically, in diurnal species, light generally increases locomotor activity (positive masking), and in nocturnal ones, it generally suppresses it (negative masking). The intergeniculate leaflet (IGL), a subdivision of the lateral geniculate complex, receives direct retinal input and is reciprocally connected with the primary circadian clock, the suprachiasmatic nucleus (SCN). Here, we evaluated the influence of the IGL on masking and the circadian system in a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), by determining the effects of bilateral IGL lesions on general activity under different lighting conditions. To examine masking responses, light or dark pulses were delivered in the dark or light phase, respectively. Light pulses at Zeitgeber time (ZT) 14 increased activity in control animals but decreased it in animals with IGL lesions. Dark pulses had no effect on controls, but significantly increased activity in lesioned animals at ZT0. Lesions also significantly increased activity, primarily during the dark phase of a 12:12 light/dark cycle, and during the subjective night when animals were kept in constant conditions. Taken together, our results suggest that the IGL plays a vital role in the maintenance of both the species-typical masking responses to light, and the circadian contribution to diurnality in grass rats

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

Suprachiasmatic Nucleus and Subparaventricular Zone Lesions Disrupt Circadian Rhythmicity but Not Light-Induced Masking Behavior in Nile Grass Rats

The ventral subparaventricular zone (vSPVZ) receives direct retinal input and influences the daily patterning of activity in rodents, making it a likely candidate for the mediation of acute behavioral responses to light (i.e., masking). We performed chemical lesions aimed at the vSPVZ of diurnal grass rats (Arvicanthis niloticus) using N-methyl-D,L-aspartic acid (NMA), a glutamate agonist. Following NMA lesions, we placed grass rats in various lighting conditions (e.g., 12:12 light-dark, constant dark, constant light); presented a series of light pulses at circadian times (CT) 6, 14, 18, and 22; and placed them in a 7-h ultradian cycle to assess behavioral masking. Extensive bilateral lesions of the vSPVZ disrupted the expression of circadian rhythms of activity and abolished the circadian modulation of masking responses to light, without affecting light-induced masking behavior per se. We also found that in diurnal grass rats, NMA was capable of destroying not only neurons of the vSPVZ but also those of the suprachiasmatic nucleus (SCN), even though excitotoxins have been ineffective at destroying cells within the SCN of nocturnal rodents. The vulnerability of the grass rat\u27s SCN to NMA toxicity raises the possibility of a difference in density of receptors for glutamate between nocturnal and diurnal species. In cases in which damage extended to the SCN, masking responses to light were present and similar to those displayed by animals with damage restricted to the vSPVZ. Thus, extensive bilateral lesions of the SCN and vSPVZ disrupted the expression of circadian rhythms without affecting acute responses to light in a diurnal species. Our present and previous results suggest that retinorecipient brain areas other than the SCN or vSPVZ, such as the intergeniculate leaflet or olivary pretectal nucleus, may be responsible for the mediation of masking responses to light in the diurnal grass rat

Circadian regulation of daily rhythms in orexinergic neurons in diurnal and nocturnal rodents.

El trabajo que se presenta aquí se centra en la regulación diferencial que ejerce el sistema nervioso central sobre ritmos circadianos en una especie diurna, Arvicanthis niloticus, o rata Nile grass y una especie nocturna, Rattus norvegicus, o rata de laboratorio. En Nile grass, las neuronas que expresan orexina (ORX) mostraron un ritmo endógeno diario en la expresión de Fos, ritmo que correlaciona con el ciclo de sueño y vigilia de esta especie y que es opuesto en comparación con el ritmo visto en ratas de laboratorio. En Nile grass las neuronas de ORX reciben proyecciones sustanciales desde neuronas del núcleo supraquiasmático (SCN) que expresan el péptico vasoactivo intestinal (VIP). En contraste, en ratas de laboratorio se encontraron muy pocas fibras positivas para VIP adyacentes a neuronas de ORX. Esta diferencia entre especies sugiere un control directo por parte del SCN sobre neuronas que expresan ORX en Nile grass y una regulación más indirecta en ratas de laboratorio. Estos resultados son consistentes con la hipótesis según la cual las diferencias entre especies diurnas y nocturnas se deben a diferencias en las funciones de regiones que reciben eferencias del SCN tales como las neuronas de ORX y el hipotálamos dorsomedial (DMH

Evidence for different thermal ecotypes in range centre and trailing edge kelp populations

Determining and predicting species’ responses to climate change is a fundamental goal of contemporary ecology. When interpreting responses to warming species are often treated as a single physiological unit with a single species-wide thermal niche. This assumes that trailing edge populations are most vulnerable to warming, as it is here where a species’ thermal niche will be exceeded first. Local adaptation can, however, result in narrower thermal tolerance limits for local populations, so that similar relative increases in temperature can exceed local niches throughout a species range. We used a combination of common garden temperature heat-shock experiments (8–32 °C) and population genetics (microsatellites) to identify thermal ecotypes of northeast Atlantic range centre and trailing edge populations of the habitat-forming kelp, Laminaria digitata. Using upregulation of hsp70 as an indicator of thermal stress, we found that trailing edge populations were better equipped to tolerate acute temperature shocks. This pattern was consistent across seasons, indicating that between-population variability is fixed. High genetic structuring was also observed, with range centre and trailing edge populations representing highly distinct clusters with little gene flow between regions. Taken together, this suggests the presence of distinct thermal ecotypes for L. digitata, which may mean responses to future warming are more complex than linear range contractions. © 2019 Elsevier B.V

Normal Behavioral Responses to Light and Darkness and the Pupillary Light Reflex are Dependent Upon the Olivary Pretectal Nucleus in the Diurnal Nile Grass Rat

The olivary pretectal nucleus (OPT) is a midbrain structure that receives reciprocal bilateral retinal projections, is involved in the pupillary light reflex, and connects reciprocally with the intergeniculate leaflet (IGL), a retinorecipient brain region that mediates behavioral responses to light pulses (i.e., masking) in diurnal Nile grass rats. Here, we lesioned the OPT and evaluated behavioral responses in grass rats to various lighting conditions, as well as their anxiety-like responses to light exposure. While control grass rats remained diurnal, grass rats with OPT lesions exhibited a more night-active pattern under 12h:12h light-dark (LD) conditions. However, when placed in constant darkness, OPT-lesioned grass rats became more active during their subjective day, suggesting that an exaggerated masking response to light may be responsible for the effect of OPT lesions on locomotor activity in LD. To test this hypothesis, we presented dark and light pulses to controls and grass rats with OPT lesions; controls increased their activity in response to light, whereas those with OPT lesions significantly increased activity in response to darkness. Further, when placed in a 7-h ultradian LD cycle, animals with OPT lesions were more active during darkness than controls. OPT lesions also abolished the pupillary light reflex, but did not affect anxiety-like behaviors. Finally, in animals with OPT lesions, light did not induce Fos expression in the ventrolateral geniculate nucleus, as it did in controls. Altogether, these results suggest that masking responses to light and darkness are dependent upon nuclei within the subcortical visual shell in grass rats

Genome-wide detection of a TFIID localization element from an initial human disease mutation

Eukaryotic core promoters are often characterized by the presence of consensus motifs such as the TATA box or initiator elements, which attract and direct the transcriptional machinery to the transcription start site. However, many human promoters have none of the known core promoter motifs, suggesting that undiscovered promoter motifs exist in the genome. We previously identified a mutation in the human Ankyrin-1 (ANK-1) promoter that causes the disease ankyrin-deficient Hereditary Spherocytosis (HS). Although the ANK-1 promoter is CpG rich, no discernable basal promoter elements had been identified. We showed that the HS mutation disrupted the binding of the transcription factor TFIID, the major component of the pre-initiation complex. We hypothesized that the mutation identified a candidate promoter element with a more widespread role in gene regulation. We examined 17 181 human promoters for the experimentally validated binding site, called the TFIID localization sequence (DLS) and found three times as many promoters containing DLS than TATA motifs. Mutational analyses of DLS sequences confirmed their functional significance, as did the addition of a DLS site to a minimal Sp1 promoter. Our results demonstrate that novel promoter elements can be identified on a genome-wide scale through observations of regulatory disruptions that cause human disease