This study examined the properties of the CNS Tb regulator that change in hypoxia. Central regulation of Tb remains operational in hypoxia, however, low oxygen progressively reduces Tth as well as suppressing metabolic thermosensitivity. Combined, this suggests that the hypoxic thermoregulatory response results from a regulated decline in body temperature, similar to that observed during sleep states, torpor, and hibernation.
Steady state responses to hypoxia
Acute, but prolonged (i.e. > 2 h) exposure to hypoxia leads to major adjustments in mammalian cardiorespiratory, metabolic and thermoregulatory homeostasis (Powell et al. 1998; Mortola & Frappell, 2000). The results of the present study are no exception (Fig. 1). The overall response to hypoxia that occurred in these two ground squirrel species was: (1) a progressive reduction in Tb which is accompanied by a reduction in metabolic heat production, (2) a ventilatory stimulation (increased ), driven initially by an increase in heart rate, and later on by changes in tidal volume, and (3) a hypoxic tachycardia in mild hypoxia, that converted to a bradycardia as the metabolic requirements dropped in extreme hypoxia. The overall responses support the idea that hypoxia initiates a chemoreceptor-driven ventilatory stimulation and a decline in metabolic heat production accompanied by a fall in Tb. Ultimately, this cascade of events is geared toward the balance of oxygen supply and demand through changes in delivery (cardiorespiratory) and consumption (metabolic) pathways. This physiological response, often termed hypoxic-anapyrexia (Steiner & Branco, 2002; Steiner et al. 2002), was relatively stable throughout the experimental period, allowing for the manipulation of Th. In order to ascertain whether the hypoxic decline in Tb resulted from a regulated decrease, it was necessary to manipulate Th under these steady state conditions and observe whether concomitant changes in physiological responses reflected attempts to defend homeostatic states.
Hypothalamic control of physiological responses in hypoxia
The most profound results from the hypothalamic manipulation trials were manifested in the metabolic changes. Cooling the hypothalamus led to activation of thermogenic mechanisms proportional to the degree of cooling. This has been observed in prior work on a range of mammals from mice, rats, cats, dogs and seals (Simon et al. 1986). The proportionality of the central mechanisms for heat production are thought to reflect the presence of cold-responsive neurons (or, more specifically, from inhibitory warm-sensitive neurons) within the CNS regulator, whose output is coupled to thermogenic activation in the periphery (Bligh, 1998). The metabolic thermosensitivity is reduced, but not fully eliminated by hypoxic exposure; α values decrease 5.4- and 8.7-fold, respectively in CGS and GMGS comparing 21 to 7% O2. Shivering can still be recruited in hypoxia by hypothalamic cooling, albeit at reduced levels (Fig. 5). This alone demonstrates that the hypoxic decline in Tb is supported by thermogenic mechanisms acting with reduced amplitude (Barros et al. 2001). For the most part, the proportionality constants for cooling in other physiological parameters (e.g. fH, fR) also exhibited similar declines in hypoxia. We did not record major physiological changes that reflected regulatory responses to hypothalamic warming (in normoxia or hypoxia); ideally, however, these experiments need to be conducted in a species that exhibits profound thermolytic responses to hypothalamic heating (Parmeggiani et al. 1973) to resolve this question. An important caveat from this study, however, is that other thermoeffectors may not exhibit similar thresholds for activation in hypoxia, since recent evidence points toward multiple, independent temperature sensors and pathways within the brain responsible for activating heat loss, heat production and temperature-seeking behaviour (Nagashima et al. 2000).
Hypoxic thermoregulatory threshold and thermosensitivity
The ultimate goal of this study was to shed light on whether hypoxia induces a decrease in hypothalamic ‘set-point’ for Tb regulation, as hypothesised by Wood (1991) . The term ‘set-point’, however, has produced much debate, confusion and controversy, and recently inspired abandonment (Romanovsky, 2004; Cabanac, 2006). Part of the debate stems from different uses of the term (Cabanac, 2006), as well as from an expectation that a stable ‘reference’ neuron must exist that maintains a constant firing rate across a range of brain temperatures. Hammel's early neuron network model (Hammel, 1968, 1972), however, did not require constancy in a reference neuron, but simply a network of interacting neurons with differing thermal sensitivities (Bligh, 1998; Cabanac, 2006). In fact, simple changes in the pattern of neuronal firing (see Hori et al. 1987, 1988; Koga et al. 1987) would help explain how the hypothalamic threshold actually varies; changes in physiological state, such as fever, exercise, sleep and hibernation, may alter the inherent thermosensitivities of the neurons within the network, and thereby alter the balance point (Romanovsky, 2004, 2007) that represents the regulated Tb. We have used the term Tth similarly to Heller & Hammel (1972), as a threshold temperature that activates/deactivates the intrinsic thermosensitivity of the central Tb regulator.
Indeed, one of the major results from the current study is that the Tth is reset progressively to lower levels by hypoxic exposure. The notion that Tb is down-regulated has been a prevailing hypothesis in the literature on hypoxic anapyrexia (Kozak, 1997; Barros et al. 2001; Tattersall & Milsom, 2003). However, to date, the most compelling evidence for this assertion has come from work done on ectotherms, which actively select lower environmental temperatures in hypoxia (Dupré & Wood, 1988; Wood & Malvin, 1993; Tattersall & Boutilier, 1997; Bicego et al. 2006; Cadena & Tattersall, 2009). An active preference for lower Tb requires neurophysiological coordination, and thus strongly implies that the central thermoregulatory mechanisms are reset, since effectors for Tb control include behavioural as well as physiological mechanisms. Lowered temperature preferences have also been observed in endotherms (Gordon & Fogelson, 1991; Wood, 1995; Gordon, 1997) as well as a wide range of animals (Wood & Gonzales, 1996), suggesting an adaptive value to lowering Tb during hypoxic stress. Therefore, if hypoxia down-regulated Tb, then it should follow that correlates of energy metabolism, thermoregulation and heat loss would operate in a coordinated fashion to facilitate this drop in Tb. Further indirect evidence for hypoxia eliciting apparent declines in regulated Tb are found in cats and rabbits where artificially raising Tb back to normothermic temperatures elicits a heat stress or relative hyperthermic responses (Rohlicek et al. 1996; Seifert et al. 2006). Furthermore, changes to the thermosensitivity of hypothalamic neurons (both warm- and cold-responsive neurons) do occur (Koga et al. 1987; Tamaki & Nakayama, 1987) in hypoxia, suggesting that the hypoxic effects on α may be a direct effect on neuronal activity in the hypothalamus, as has been implied for osmotic and cardiovascular influences on Tb (Hori et al. 1987, 1988).
It is also possible, however, that the decline in α in hypoxia is a passive consequence of the drop in Tb itself, due to inherent thermal effects on neuronal firing rates (Heller & Colliver, 1974; Heller et al. 1974). We can address this by examining the temperature sensitivity for α; the relationship between α and Tth yielded Q10 values ranging from 10 to 12, substantially higher than that expected from simple temperature effects, as well as higher than that observed in hibernation (Florant & Heller, 1977; Florant et al. 1978), where Tth has been well established to be co-ordinately lowered. Indeed, the likely explanation is that the central thermosensitivity changes in hypoxia result from a direct suppressive effect on thermosensitive neuronal activity (or an alteration in the balance of excitatory and inhibitory inputs), rather than α values passively following Tb. One consequence of a suppression of neuronal activity is that the hypoxic decline in Tb produces a more variable thermoregulatory pattern due to the reduced gain or sensitivity of the CNS Tb regulator. Precedence for this is observed in ectothermic studies showing a decreased precision of regulated Tb in hypoxia (Cadena & Tattersall, 2009), as well as studies in mammals that demonstrate that the hypoxic decline in Tb is more variable and also strongly dependent on ambient temperature (Barros et al. 2001; Bishop et al. 2001; Levesque & Tattersall, 2009). Indeed, one interesting result from the present study is the remarkable similarity between the changes in α and Tth and those observed during different sleep states in rodents. During slow wave sleep (SWS), α and Tth are partially reduced, whereas during REM sleep, Tth and α are entirely absent (Glotzbach & Heller, 1975, 1976; Sakaguchi et al. 1979), which has led to the notion that REM sleep is essentially a period where thermoregulatory defences are abandoned. Hypoxia appears to produce a qualitatively similar state to SWS; coincidentally, hypoxia also produces alterations in EEG patterns, which show a relative increase in slow wave activity. These similar patterns suggest parallel mechanisms between SWS and hypoxia, although whether hypoxia influences the hypothalamic regulator via similar processes is unknown.
Under resting, normoxic conditions, feedback from extrahypothalamic warming would appear to be minor (Heller et al. 1974) since Tb continues to rise linearly during Th cooling without abatement. Indeed, ground squirrels exhibit little inhibitory extra-hypothalamic feedback during euthermia and hibernation (Heller & Hammel, 1972). In hypoxia, the relative amount of extrahypothalamic sensitivity (assessed by the heat gain ratio) trended toward zero, since cooling Th, which augments heat production, does not evoke the same rise in Tb as it did in normoxia. This may actually be the result of a sustained peripheral vasodilatation in hypoxia (Tattersall & Milsom, 2003), allowing the centrally derived heat to be driven from the animals, or from the involvement of peripheral chemoreceptor activation acting via medullary pathways producing a sustained reduction in sympathetic nervous activation of thermogenesis (Madden & Morrison, 2005). Combined with the low hypothalamic thermosensitivity, this may explain why hypoxia also produces an ambient-temperature-dependent Tb in addition to lowering Tth (Barros et al. 2001). Similarly, we observed that was directly related to oxygen level, indicating that the threshold for heat activation is actually below the apparent steady state value in hypoxia. Thus, despite an achievement of steady state physiological responses to hypoxia, Tth drops below and in advance of Th, similar to what is observed during entrance into hibernation (Heller et al. 1977). Furthermore, we also observed instances in hypoxic squirrels where Th did not return to previous values after completion of Th manipulation (Fig. 7), reinforcing that the threshold to activate thermogenesis in hypoxia is very often well below that of Th.
Interestingly, upon initial exposure to hypoxia, Th−Tb, which is normally positive, decreases, implying that the stimulus to regulate an elevated Th is diminished. After 2 h of exposure to hypoxia, however, this difference begins to rise, driven by a stabilisation of Th (Fig. 2). This suggests that Th is being regulated in hypoxia; upon re-oxygenation, the Th−Tb difference rapidly rises, for, perhaps two reasons. Firstly, Tth for thermogenesis would instantly return to normal values (as witnessed by the near-instantaneous activation of shivering upon returning oxygen levels to 21%; Fig. 6), and given the small size of the brain relative to its high blood flow, maximal thermogenesis would raise Th prior to Tb. It is also possible that the greater difference between Th and Tb at this point is due to selective brain warming, which is consistent with how many hibernators re-warm from torpor; an elevated thoracic and head temperature is achieved prior to re-warming the rest of the body, achieved via sympathetically mediated constriction in the periphery (Osborne et al. 2005).
Universality of the hypoxic thermoregulatory response?
Although the present study demonstrated a robust decline in Tb, interestingly, acute hypoxia does not exert profound changes in Tb or in larger mammals (>2 kg; Frappell et al. 1992), which may be due to a size-limited inability to cool significantly, akin to the size constraints of deep hibernation observed in small mammals (Geiser, 1998). Given, however, that the lowering of Tb is a nearly ubiquitous response to hypoxia in the animal kingdom, large mammals may still exhibit the appropriate control mechanisms for reducing Tb in hypoxia, but are simply incapable of doing so due to size restrictions. Equally, however, the slight decline in Tb that is observed in large mammals in hypoxia may be due to scaling influences on hypothalamic thermosensitivity. Mammalian hypothalamic thermosensitivity scales negatively (exponent =−0.37) with body mass (Heller, 1978), suggesting a diminishing importance of central neuronal thermosensitivity in maintaining Tb in large animals. If hypoxia reduces primarily central thermosensitivity, with minimal effects on peripheral thermosensation, this may explain the profound reduction in Tb of small mammals. Further examination of central and peripheral thermosensitivities in hypoxic states is warranted to test this hypothesis.
Conclusions and perspectives
The present study illustrates that informative features of how the CNS Tb regulator operates can be elucidated from simple changes in inspired oxygen. This approach has shed light on a fundamental homeostatic mechanism (temperature regulation), demonstrating that a change in oxygen, which could be manifested in a number of ways (e.g. alteration in brain blood flow, airway occlusion/asphyxia, sleep apnoea, as well as naturally hypoxic states), produces a rheostatic adjustment in homeostasis (Mrosovsky, 1990). Indeed, Tb regulation is lowered from ∼38°C in normoxia to ∼30°C in hypoxia. Central thermoregulatory control, however, is still intact in hypoxia, although the thresholds for changes in metabolism and shivering are reduced in magnitude. The degree of central thermosensitivity is drastically reduced, in a manner similar, but more profound, to that observed during SWS. Thus, although hypoxia produces a decrease in the regulated Tb, this begs the question about whether the term ‘hypoxia-induced anapyrexia’ (Steiner & Branco, 2002; Steiner et al. 2002) is entirely appropriate to apply to a state that reduces both the regulated temperature and the sensitivity (α) of the regulator itself. Equally, however, hypoxia does not produce a state where thermoregulatory responses are completely abandoned, arguing against ‘hypothermia’ being an appropriate description of the hypoxic thermoregulatory response.