Mature Naked Tj !!EXCLUSIVE!!
Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals.
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Consistent with this environmental challenge and a long subterranean evolutionary history dating to the Miocene , naked mole-rats display several physiological adaptations for survival in a chronically hypoxic environment. Notably, their hemoglobin has a higher affinity for oxygen than most other mammals , and their weight-specific metabolic rate is about one-third less than that of other rodents . We recently reported another characteristic consistent with evolving in a hypoxic environment : We found that hippocampal brain slices from adult naked mole-rats maintained synaptic transmission at low oxygen concentrations that caused transmission to decrease or cease altogether in slices from laboratory mice. Also, in nominally zero oxygen, naked mole-rat slices maintained electrophysiological function more than three times as long as slices from mice, and frequently recovered even after an anoxic depolarization lasting several minutes.
In one set of experiments, slices were challenged by perfusion of ACSF containing elevated concentrations of potassium. Baseline images were recorded every 20 seconds for 20 minutes prior to application of potassium to ensure that the slice was healthy and that movement was negligible. For potassium application, the bath solution was switched to one with a high concentration of potassium (equimolar replacement of NaCl with KCl). In pilot tests with potassium concentrations ranging from 5 to 30 mM, we found that 15 and 25 mM were well on the dynamic part of the concentration/response curve for slices from P6 mice (which had the maximum response) and did not saturate the fura-2 indicator response. Based on that, we collected a complete data set for both 15 and 25 mM potassium. Images were collected every 20 seconds for 15 minutes beginning 1 or 4 minutes before switching to one of the high potassium solutions. After 2 minutes, the solution was switched back to normal bath solution and the slice was allowed to recover. Ten minutes after recovery, the other high potassium solution was applied for 2 minutes followed by recovery. The order of potassium solutions was alternated between slices. Pseudocolor example images of fluorescence at 380 nm taken before, during, and after application of 25 mM potassium are shown in Figure 1E, F, G for a P6 mouse and I, J, K for a P6 naked mole-rat. The curves showing ratiometric data for all 15 minutes of testing are shown in Figure 1H and L. Note that in these particular example slices, we collected data for 4 minutes prior to switching to a high potassium solution. This duration of baseline illustrates the stability of the response. Data collected for group analyses used a baseline duration of 1 minute.
A. Data from P6 (9 slices, 4 animals) and P20 (8 slices, 5 animals) mouse hippocampal slices. Images were collected over 15 minutes. The black bars indicate the 2 minute time course that 25 mM potassium bath solution was applied. B. Data from P6 (10 slices, 4 animals) and P20 (15 slices, 4 animals) naked mole-rat slices tested under the same conditions as A. C. Summary data showing the change in maximal calcium with age for P6 and P20 mice and naked mole-rats during the 2 minute exposure to 25 mM K+. D. Summary data showing the change in maximal calcium with age for P6 and P20 mice and naked mole-rats during a 2 minute exposure to 15 mM K+. * and ** correspond to significance at p
There are several noteworthy aspects of these data. The potassium challenge demonstrated a concentration-dependent increase in fluorescence that was greater in some cases than the responses due to hypoxia, suggesting that the hypoxia measurements were well within the dynamic range of the fura-2 indicator. In addition, the responses to potassium challenge were much more similar for naked mole-rats and mice than the responses to hypoxia; this was particularly evident for the older age groups where the potassium responses were not significantly different. This is important because it shows that the blunted calcium response to hypoxia in naked mole-rats cannot be entirely accounted for by a generalized reduction in responsiveness to all stimuli. Furthermore, it suggests that the differences we found were not due to species differences in dye uptake which in our experience is most problematic in older mice.
Finally, the older age groups had a smaller calcium accumulation response to potassium challenge than the younger age groups in both mouse and naked mole-rat, which is the opposite pattern from the one we observed with hypoxia. This is important because it suggests that the age differences we found for hypoxia were not due to poor slice health in older animals.
The main finding of this study is that hippocampal neurons in naked mole-rats show a blunted intracellular calcium response to hypoxia when compared to neurons in mice. The attenuated calcium accumulation response in naked mole-rat hippocampus was highly significant compared to the response in mouse hippocampus, whether assessed in animals at an early stage of postnatal development (P6) or in weanlings (P20). Technical issues did not permit a comparison of calcium responses in adult mice and naked mole-rats because fura-2 AM staining of adult tissue is impaired as the neuropil develops; however a previous electrophysiological study demonstrated that adult (>1 year old) naked mole-rat hippocampus is extremely tolerant to two related effects of hypoxia: namely, the suppression of synaptic transmission during partial hypoxia and the collapse of membrane potentials (anoxic depolarization) that accompanies severe oxygen deprivation .
The attenuation of calcium response in naked mole-rat hippocampus appears to be specifically related to hypoxia tolerance because the calcium response to potassium challenge was much more similar for naked mole-rats and mice. First, both mice and naked mole-rat neurons showed an age-dependent reduction in calcium response to potassium rather than the age-dependent increase in calcium response to hypoxia. Second, at P20, naked mole-rat neurons showed a highly significant reduction (68%) in calcium response to hypoxia compared to mouse neurons, but no significant difference in calcium response to potassium compared to mouse neurons.
Naked mole-rats share two important features with these other model systems: resistance to hypoxia in vivo and an attenuated hypoxia-induced neuronal calcium response in vitro. We do not yet know the underlying mechanism(s) behind the extreme tolerance to hypoxia in naked mole-rat neurons. However, it appears that, unlike the turtle , an increase in adenosine does not contribute to the diminished increase in calcium. In a previous study, we showed that hippocampal cells in adult naked mole-rats were less sensitive to adenosine as compared to cells from mice . We suggested that the adult naked mole-rat brain resembles the neonatal rat (and mouse) brain in terms of response to adenosine, resistance to hypoxia, and lack of paired-pulse facilitation . (The robust staining of naked mole-rat neurons with fura-2 AM at P42, an age when staining of mouse or rat neurons is poor and inconsistent, may also reflect a difference in neuronal maturation.) Furthermore, we recently showed that adult naked mole-rat brain retains more of the (neonatally abundant) NMDA receptor subunit, NR2D, compared to mice . The calcium imaging results from the present study are not inconsistent with the notion that slowed or arrested brain development may endow the naked mole-rat brain with extreme hypoxia tolerance. Even at P42 (the most advanced age tested), naked mole-rat brain showed an attenuated calcium accumulation response to hypoxia, compared to neonatal (P6) mice.
Currently there are relatively few comparative studies on intrinsic hypoxia tolerance in brain slices in mammals. However, one such study measured membrane potentials from visual cortex slices from diving seals and mice . They found that under severe hypoxia, slices from seals maintained synaptic function approximately 4 times longer than slices from mice (19 minutes versus 5 minutes). This is similar to what we found previously when comparing hippocampal slices from naked mole-rats and mice (12.63 minutes versus 2.16 minutes ). Using cell survival as a metric, another study  showed that CA1 cells in hippocampal slices from both hibernating and active 13-lined ground squirrels survived longer than CA1 cells in slices from rats. Consistent with this finding, a variety of hypoxia tolerant adaptations have been found in brain cells of hibernating species (see  for review). In our present study, we did not look at cell survival because our protocol was designed to measure recovery. However, our previous study which measured physiological responses to hypoxia clearly showed that hippocampal slices from naked mole-rats were able to survive and/or recover from hypoxia applications that slices from mice could not recover from . Non-mammalian models of hypoxia tolerance include some fishes, frogs, and turtles . Interestingly, forebrain cells from tadpoles  and cortical slices from turtles  show increases in internal calcium during severe but survivable hypoxia without showing the cell damage characteristic of mammalian neurons exposed to high calcium. 041b061a72