A stereotaxic atlas of the brain of the naked mole-rat (Heterocephalus glaber)

The naked mole-rat (Rodentia, Bathyergidae: Heterocephalus glaber) is a strictly subterranean eusocial mammal. These rodents show a suite of morphological and physiological adaptations, including brain specializations, to this underground milieu that they have inhabited since the early Miocene. Recently, naked mole-rats have received considerable attention as the longest living rodent known, and some of these brain specializations may be potentially important to their exceptional longevity. To serve as a basis for future brain studies, we have constructed a stereotaxic atlas of the brain of this species, labeling all major brain structures.     

Catecholaminergic innervation of interscapular brown adipose tissue in the naked mole-rat (Heterocephalus glaber)

The thermogenic potential of the interscapular brown fat pad in the naked mole-rat Heterocephalus glaber , that exhibits poikilothermic thermal responses to changing temperatures is reported. Histological and ultrastructural study of the brown fat pad showed that it consists of layers of skeletal muscle interposed between the layers of brown adipose tissue with both unilocular and multilocular adipocytes. Large numbers of mitochondria were present between and around the lipid droplets of these cells. Glyoxylic acid condensation, used to demonstrate catecholaminergic nerves, was evident in low concentrations in the connective tissue between the brown adipocytes. A 3-dimensional computer-aided reconstruction of the fat pad showed the extent and ramification of nerves and blood vessels between the adipocytes. These findings show that although the naked mole-rat is regarded as an endothermic poikilotherm, it possesses anatomical features usually found in homeothermic mammals, which are essential for thermogenesis.     

Circadian rhythms of locomotor activity in naked mole-rats (Heterocephalus glaber)

A wide variety of organisms exhibit various circadian rhythms in their behavior and physiology. Circadian rhythms are regulated by internal clocks that are generally entrained primarily by the environmental light:dark (L:D) cycle. There have been few studies of circadian rhythms in fossorial species that inhabit an environment where day-night variations are minimal and where exposure to light occurs infrequently. In this study, circadian patterns of wheel-running activity were examined in naked mole-rats (Heterocephalus glaber). Naked mole-rats are fossorial and eusocial, living in colonies of 60-70 animals with only one breeding female. Most individual mole-rats that ran on wheels (65%) exhibited robust circadian rhythms of locomotor activity, entrained to various L:D cycles, and free-ran in constant darkness (DD) with taus averaging 23.5 h. The remainder of the animals either free-ran or were arrhythmic under the various L:D cycles. Mole-rats generally failed to entrain to non-24-h T-cycles with period lengths ranging from T=23 h to T=25 h. There was considerable inter-individual variation in the circadian patterns of locomotor activity in naked mole-rats as is observed in other subterranean mammals that have been studied. In contrast to the results obtained when mole-rats were individually housed with access to running wheels, circadian rhythms of general locomotor activity were typically not observed for animals monitored while they were housed in a colony setting. However, clear nocturnal rhythms of general locomotor activity were displayed by four males while residing in their home colonies. Two of these males exhibited the physical appearance of a disperser morph - subordinate individuals that are believed to leave their home colonies to achieve reproductive opportunities elsewhere. All four of these males were among the largest males in their respective colonies. These results demonstrate that although naked mole-rats are not frequently exposed to light, the species has retained the capacity to exhibit locomotor patterns of circadian rhythmicity and has the ability to entrain to 24-h L:D cycles. The possible adaptive function of this circadian capacity is discussed.     

Postnatal development of the eye in the naked mole rat (Heterocephalus glaber)

The naked mole rat (Heterocephalus glaber) is a subterranean rodent whose eyes are thought to be visually nonfunctional and as such is an ideal animal with which to pursue questions in evolutionary developmental biology. This report is the first in-depth study on the development and morphology of the naked mole rat eye. Using standard histological analysis and scanning and transmission electron microscopy, we describe the structural features of the eye. We further report on the morphological changes that accompany the development of this eye from neonate to adult and compare them with those that occur during mouse eye development. We observed numerous abnormalities in the shape and cellular arrangement of the structures of the anterior chamber, with notable malformations of the lens. Cell proliferation and cell death assays were conducted to investigate the possible causes of lens malformation. We found that neither of these processes appeared abnormal, indicating that they were not responsible for the lens phenotype of the mole rat. In order to investigate the process of lens differentiation, we analyzed the expression of gamma-crystallins using Western blots and immunocytochemistry. At birth, levels of gamma-crystallin appear normal, but soon thereafter, the gamma-crystallin expression is terminated. Absence of detectable gamma-crystallins in adults suggests that there is a gradual degradation and loss of these proteins. The evolutionary factors that could be responsible for the eye morphology of the naked mole rat are discussed. A model for abnormal lens differentiation and the role it plays in the morphogenesis of the rest of the eye in the naked mole rats is proposed.     

Factors influencing metabolic rate in naked mole-rats (Heterocephalus glaber).

Naked mole-rats (Heterocephalus glaber) are fossorial, eusocial mammals that live in colonies averaging about 70 individuals. Metabolic regulation is of particular interest in this species because it is one of only two naturally occurring small mammals that are hairless. Further, relative to other small mammals, naked mole-rats exhibit low body temperature (Tb) and weak capacity to maintain Tb above the ambient temperature (Ta). The present study examined effects of Ta, norepinephrine (NE), and chronic food restriction on O2 consumption (as a measure of metabolism) in naked mole-rats. Studies were performed in both awake and anesthetized animals. Metabolic rate decreased with increasing T. over the range of 23-34 degrees C in awake mole-rats, whereas in anesthetized animals rates of O2 consumption were very low over this entire range of Ta and tended to increase with increasing Ta. Injections of NE led to rapid increases in metabolic rate at all Tas in anesthetized subjects and also at Ta = 34 degrees C in awake mole-rats. However, at Tas of 29 and 23 degrees C, awake subjects given NE showed little stimulation of O2 consumption beyond the already elevated baseline rates observed at these Tas. During chronic restriction of food to 60-70% of their normal daily consumption mole-rats exhibited decreased rates of metabolism; metabolic rate was not altered following several hours of acute food deprivation. Food consumption remained somewhat decreased after a period of chronic food restriction, even when animals were returned to ad lib conditions. However, body weights returned to prerestriction values, despite the continued reduction in ad lib food intake. These observations suggest that mole-rats may be capable of long-lasting metabolic adaptations as a means to cope with restricted food supply. These findings are discussed in relation to adaptation of this fossorial species to a habitat where food has a patchy distribution. Naked mole-rats, with their several unusual thermoregulatory and behavioral features, provide an intriguing model for studies of mammalian metabolic regulation.     

Cortical, callosal, and thalamic connections from primary somatosensory cortex in the naked mole-rat (Heterocephalus glaber), with special emphasis on the connectivity of the incisor representation

We investigated the distribution of cortical, callosal, and thalamic connections from the primary somatosensory area (S1) in naked mole-rats, concentrating on lower incisor and forelimb representations. A neuronal tracer (WGA-HRP) was injected into the center of each respective representation under guidance from microelectrode recordings of neuronal activity. The locations of cells and terminals were determined by aligning plots of labeled cells with flattened cortical sections reacted for cytochrome oxidase. The S1 lower incisor area was found to have locally confined intrahemispheric connections and longer connections to a small cluster of cells in the presumptive secondary somatosensory (S2) and parietal ventral (PV) incisor fields. The S1 incisor area also had sparse connections with anterior cortex, in presumptive primary motor cortex. Homotopic callosal projections were identified between the S1 lower incisor areas in each hemisphere. Thalamocortical connections related to the incisor were confined to ventromedial portions of the ventral posterior medial subnucleus (VPM) and posterior medial nucleus (Po). Injections into the S1 forelimb area revealed reciprocal intrahemispheric connections to S2 and PV, to two areas in frontal cortex, and to two areas posterior to S1 that appear homologous to posterior lateral area and posterior medial area in rats. The S1 forelimb representation also had callosal projections to the contralateral S1 limb area and to contralateral S2 and PV. Thalamic distribution of label from forelimb injections included ventral portions of the ventral posterior lateral subnucleus (VPL), dorsolateral Po, the ventral lateral nucleus, and the ventral medial nucleus and neighboring intralaminar nuclei.     

Underdeveloped Extraocular Muscles in the Naked Mole‐Rat (Heterocephalus glaber)

The extraocular muscles (EOM), the effector arm of the ocular motor system, have a unique embryological origin and phenotype. The naked mole‐rat (NMR) is a subterranean rodent with an underdeveloped visual system. It has not been established if their ocular motor system is also less developed. The NMR is an ideal model to examine the potential codependence of oculomotor and visual system development and evolution. Our goal was to compare the structural features of NMR EOMs to those of the mouse, a similar sized rodent with a fully developed visual system. Perfusion‐fixed whole orbits and EOMs were dissected from adult NMR and C57BL mice and examined by light and electron microscopy. NMR orbital anatomy showed smaller EOMs in roughly the same distribution around the eye as in mouse and surrounded by a very small Harderian gland. The NMR EOMs did not appear to have the two‐layer fiber distribution seen in mouse EOMs; fibers were also significantly smaller (112.3 ± 46.2 vs. 550.7 ± 226 sq μm in mouse EOMs, *P < 0.05). Myofibrillar density was less in NMR EOMs, and triad and other membranous structures were rudimentary. Finally, mitochondrial volume density was significantly less in NMR EOMs than in mouse EOM (4.5% ± 1.9 vs. 21.2% ± 11.6, respectively, *P < 0.05). These results demonstrate that NMR EOMs are smaller and less organized than those in the mouse. The “simpler” EOM organization and structure in NMR may be explained by the poor visual ability of these rodents, initially demonstrated by their primitive visual system. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Long bone histomorphogenesis of the naked mole-rat: Histodiversity and intraspecific variation

Lacking fur, living in eusocial colonies and having the longest lifespan of any rodent, makes naked mole-rats (NMRs) rather peculiar mammals. Although they exhibit a high degree of polymorphism, skeletal plasticity and are considered a novel model to assess the effects of delayed puberty on the skeletal system, scarce information on their morphogenesis exists. Here, we examined a large ontogenetic sample (n = 76) of subordinate individuals to assess the pattern of bone growth and bone microstructure of fore- and hindlimb bones by using histomorphological techniques. Over 290 undecalcified thin cross-sections from the midshaft of the humerus, ulna, femur, and tibia from pups, juveniles and adults were analyzed with polarized light microscopy. Similar to other fossorial mammals, NMRs exhibited a systematic cortical thickening of their long bones, which clearly indicates a conserved functional adaptation to withstand the mechanical strains imposed during digging, regardless of their chisel-tooth predominance. We describe a high histodiversity of bone matrices and the formation of secondary osteons in NMRs. The bones of pups are extremely thin-walled and grow by periosteal bone formation coupled with considerable expansion of the medullary cavity, a process probably tightly regulated and adapted to optimize the amount of minerals destined for skeletal development, to thus allow the female breeder to produce a higher number of pups, as well as several litters. Subsequent cortical thickening in juveniles involves high amounts of endosteal bone apposition, which contrasts with the bone modeling of other mammals where a periosteal predominance exists. Adults have bone matrices predominantly consisting of parallel-fibered bone and lamellar bone, which indicate intermediate to slow rates of osteogenesis, as well as the development of poorly vascularized lamellar-zonal tissues separated by lines of arrested growth (LAGs) and annuli. These features reflect the low metabolism, low body temperature and slow growth rates reported for this species, as well as indicate a cyclical pattern of osteogenesis. The presence of LAGs in captive individuals was striking and indicates that postnatal osteogenesis and its consequent cortical stratification most likely represents a plesiomorphic thermometabolic strategy among endotherms which has been suggested to be regulated by endogenous rhythms. However, the generalized presence of LAGs in this and other subterranean taxa in the wild, as well as recent investigations on variability of environmental conditions in burrow systems, supports the hypothesis that underground environments experience seasonal fluctuations that may influence the postnatal osteogenesis of animals by limiting the extension of burrow systems during the unfavorable dry seasons and therefore the finding of food resources. Additionally, the intraspecific variation found in the formation of bone tissue matrices and vascularization suggested a high degree of developmental plasticity in NMRs, which may help explaining the polymorphism reported for this species. The results obtained here represent a valuable contribution to understanding the relationship of several aspects involved in the morphogenesis of the skeletal system of a mammal with extraordinary adaptations.     

Antioxidants do not explain the disparate longevity between mice and the longest-living rodent, the naked mole-rat

The maximum lifespan of naked mole-rats (NMRs; Heterocephalus glaber) is greater than that of any other rodent. These hystricognaths survive in captivity >28 years, eight-times longer than similar-sized mice. The present study tested if NMRs possess superior antioxidant defenses compared to mice and if age-related interspecies changes in antioxidants were evident. Activities of Cu/Zn superoxide dismutase (Cu/Zn, SOD), Mn SOD, catalase and cellular glutathione peroxidase (cGPx) were measured in livers of physiologically equivalent age-matched NMRs (30, 75 and 130 months) and CB6F1 mice (4, 12 and 18 months). In mice, Mn SOD activity increased with age, while the activity of catalase and cGPx declined. None of the antioxidants changed with age in mole-rats. cGPx activity of NMRs was 70-times lower (p < 0.0001) than in mice, and resembled that of cGPx knock-out animals. NMRs may partially compensate for the lower cGPx when compared to mice, by having moderately higher activities of the other antioxidants. It is nonetheless unlikely that antioxidant defenses are responsible for the eight-fold longevity difference between these two species. Maintenance of constant antioxidant defenses with age in NMRs concurs with previous physiological data, suggesting delayed aging in this species.     

Bone remodeling in the longest living rodent,the naked mole-rat: Interelement variation and the effects of reproduction

The pattern of bone remodeling of one of the most peculiar mammals in the world, the naked mole-rat (NMR), was assessed. NMRs are known for their long lifespans among rodents and for having low metabolic rates. We assessed long-term in vivo bone labeling of subordinate individuals, as well as the patterns of bone resorption and bone remodeling in a large sample including reproductive and non-reproductive individuals (n = 70). Over 268 undecalcified thin cross-sections from the midshaft of humerus, ulna, femur and tibia were analyzed with confocal fluorescence and polarized light microscopy. Fluorochrome analysis revealed low osteogenesis, scarce bone resorption and infrequent formation of secondary osteons (Haversian systems) (i.e., slow bone turnover), thus most likely reflecting the low metabolic rates of this species. Secondary osteons occurred regardless of reproductive status. However, considerable differences in the degree of bone remodeling were found between breeders and non-breeders. Pre-reproductive stages (subordinates) exhibited quite stable skeletal homeostasis and bone structure, although the attainment of sexual maturity and beginning of reproductive cycles in female breeders triggered a series of anabolic and catabolic processes that up-regulate bone turnover, most likely associated with the increased metabolic rates of reproduction. Furthermore, bone remodeling was more frequently found in stylopodial elements compared to zeugopodial elements. Despite the limited bone remodeling observed in NMRs, the variation in the pattern of skeletal homeostasis (interelement variation) reported here represents an important aspect to understand the skeletal dynamics of a small mammal with low metabolic rates. Given the relevance of the remodeling process among mammals, this study also permitted the comparison of such process with the well-documented histomorphology of extinct therapsids (i.e., mammalian precursors), thus evidencing that bone remodeling and its endocortical compartmentalization represent ancestral features among the lineage that gave rise to mammals. It is concluded that other factors associated with development (and not uniquely related to biomechanical loading) can also have an important role in the development of bone remodeling.     

Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease

Amyloid beta (Aβ) is implicated in Alzheimer's disease (AD) as an integral component of both neural toxicity and plaque formation. Brains of the longest-lived rodents, naked mole-rats (NMRs) approximately 32 years of age, had levels of Aβ similar to those of the 3xTg-AD mouse model of AD. Interestingly, there was no evidence of extracellular plaques, nor was there an age-related increase in Aβ levels in the individuals examined (2–20+ years). The NMR Aβ peptide showed greater homology to the human sequence than to the mouse sequence, differing by only 1 amino acid from the former. This subtle difference led to interspecies differences in aggregation propensity but not neurotoxicity; NMR Aβ was less prone to aggregation than human Aβ. Nevertheless, both NMR and human Aβ were equally toxic to mouse hippocampal neurons, suggesting that Aβ neurotoxicity and aggregation properties were not coupled. Understanding how NMRs acquire and tolerate high levels of Aβ with no plaque formation could provide useful insights into AD, and may elucidate protective mechanisms that delay AD progression.     

Adult naked mole-rat brain retains the NMDA receptor subunit GluN2D associated with hypoxia tolerance in neonatal mammals

Adult naked mole-rats show a number of systemic adaptations to a crowded underground habitat that is low in oxygen and high in carbon dioxide. Remarkably, brain slice tissue from adult naked mole-rats also is extremely tolerant to oxygen deprivation as indicated by maintenance of synaptic transmission under hypoxic conditions as well as by a delayed neuronal depolarization during anoxia. These characteristics resemble hypoxia tolerance in brain slices from neonates in a variety of mammal species. An important component of neonatal tolerance to hypoxia involves the subunit composition of NMDA receptors. Neonates have a high proportion of NMDA receptors with GluN2D subunits which are protective because they retard calcium entry into neurons during hypoxic episodes. Therefore, we hypothesized that adult naked mole-rats retain a protective, neonatal-like, NMDA receptor subunit profile. We used immunoblotting to assess age-related changes in NMDA receptor subunits in naked mole-rats and mice. The results show that adult naked mole-rat brain retains a much greater proportion of the hypoxia-protective GluN2D subunit compared to adult mice. However, age-related changes in other subunits (GluN2A and GluN2B) from the neonatal period to adulthood were comparable in mice and naked mole-rats. Hence, adult naked mole-rat brain only retains the neonatal NMDA receptor subunit that is associated with hypoxia tolerance.     

The energetics of huddling in two species of mole-rat (Rodentia: Bathyergidae)

Small rodents with a large surface-area-to-volume ratio and a high thermal conductance are likely to experience conditions where they have to expend large amounts of energy in order to maintain a constant body temperature at low ambient temperatures. The survival of small rodents is thus dependent on their ability to reduce heat loss and increase heat production at low ambient temperatures. Two such animals are the social subterranean rodents Cryptomys damarensis (the Damaraland mole-rat) and Cryptomys hottentotus natalensis (the Natal mole-rat). This study examined the energy savings associated with huddling as a behavioural thermoregulatory mechanism to conserve energy in both these species. Individual oxygen consumption (VO(2)) was measured in groups ranging in size from one to 15 huddling animals for both species at ambient temperatures of 14, 18, 22, 26 and 30 degrees C. Savings in energy (VO(2)) were then compared between the two species. Significant differences in VO(2) (p<0.05) were found within each species, indicating that both Damaraland mole-rats and Natal mole-rats saved more energy in larger as opposed to smaller groups. VO(2) was also different between the two species, with Damaraland mole-rats showing a higher decrease in VO(2) with increasing group size compared to Natal mole-rats. These findings suggest that huddling confers significant energy savings in both species and that the amount of energy saved is related to each species' ecology. More generally, these findings suggest that group living desert-adapted species are likely to be more prone to heat loss at low ambient temperatures than temperate-adapted species, especially at low group sizes. This is presumably offset against the advantages obtained by having a low metabolic rate and avoiding hyperthermia when temperatures are hot.     

c-Fos expression in the visual system of the tree shrew (Tupaia belangeri)

c-Fos is a nuclear phosphoprotein coded by the proto-oncogen c-fos which can be detected immunohistochemically after both physiological and pathological stimuli. This property is of great importance, because it offers a valuable tool for morphofunctional identification of activated neurons. We have studied the neuronal activity in the visual pathway of Tupaia belangeri within the following anatomical structures: retina, superior colliculus (SC), dorsal lateral geniculate nucleus (dLGN), pulvinar (Pu), parabigeminal (PBG) nucleus and primary visual cortex (V1) analyzing the c-Fos expression after exposing the tree shrews to different light stimuli (white light –control positive group–, green light, blue light and darkness conditions –control negative group–). Our findings suggest that in the retina, the ganglion cells and the cells of the inner nuclear layer respond better to blue and green light stimuli, when comparing the c-Fos expression between white, green, blue lights and darkness conditions. However, in the SC, dLGN, Pu, PBG nucleus and V1 another pattern of c-Fos expression is observed: a maximum expression for the control positive group, a minimum expression for the control negative group and intermediate expressions within the blue and green light groups. Conclusion: the expression levels of c-Fos protein are able to show significant differences between distinct light stimuli in all anatomical structures studied (retina, SC, dLGN, Pu, PBG and V1) of T. belangeri.     

Complex evolution of X and Y autosomal translocations in the giant mole-rat, Cryptomys mechowi (Bathyergidae)

Cross-species chromosome painting was used to determine homologous chromosomal regions between two species of mole-rat, the naked mole-rat, Heterocephalus glaber (2n = 60), and the giant mole-rat, Cryptomys mechowi (2n = 40), using flow-sorted painting probes representative of all but two of the H. glaber chromosomal complement. In total 43 homologous regions were identified in the C. mechowi genome. Eight H. glaber chromosomes are retained in toto in C. mechowi, and 13 produce two or more signals in this species. The most striking difference in the karyotypes of the two taxa concerns their sex chromosomes. The H. glaber painting probes identified a complex series of translocations that involved the fractionation of four autosomes and the subsequent translocation of segments to the sex chromosomes and to autosomal partners in the C. mechowi genome. An intercalary heterochromatic block (IHB) was detected in sex chromosomes of C. mechowi at the boundary with the translocated autosomal segment. We discuss the likely sequence of evolutionary events that has led to the contemporary composition of the C. mechowi sex chromosomes, and consider these in the light of prevailing views on the genesis of sex chromosomes in mammals.     

Central nervous system neuroblastoma in a wild deer (Capreolus capreolus)

This report describes a central nervous system (CNS) neuroblastoma in a wild deer (Capreolus capreolus) of approximate age 10 years. The doe was found in a barn with a fractured right forelimb and was shot. The animal was submitted for post-mortem examination in order to exclude rabies. The tumour was located in the midline of the mesencephalon with replacement of thalamus and parts of the hypothalamus and infiltration into the adjacent neuroparenchyma and the right lateral and third ventricles. Microscopically, the neoplastic cells were arranged in sheets and nests forming Homer-Wright- and pseudorosettes. Immunohistochemically, the tumour cells expressed neuron-specific enolase, synaptophysin, doublecortin and neurofilament. This is the first report of an infiltrative CNS neuroblastoma in a wild deer.     

Zebrafish R-cadherin (Cdh4) controls visual system development and differentiation.

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization.     

Central nervous system development in rabbits (Oryctolagus cuniculus L. 1758)

The present study describes the embryonic and fetal development of the central nervous system in rabbits from the seventh day after conception until the end of the full-term fetal period. A total of 19 embryonic and fetal samples were carefully dissected and microscopically analyzed. Neural tube closure was observed between 7.5 and 8 days of gestation. Primordial encephalic vesicle differentiation and spinal canal delimitation were observed on the 12th day of gestation. Histologically, on the 15th day of gestation, the brain, cerebellum, and brain stem were delimited. On the 18th day of gestation, the cervical and lumbar intumescences of the spinal cord were visible. On the 28th day of gestation, four-cell layers could be distinguished in the cerebral cortex, while the cerebellar cortex was still differentiating. Overall, the morphological aspects of the embryonic and fetal developmental phases in rabbits were highly similar to those in humans. Thus, the present study provides relevant information highlighting rabbits as an excellent candidate animal model for preclinical research on human neurological diseases given the high adaptability of rabbits to bioterium conditions and the similarity of morphological events between rabbits and humans.