Mitochondrial respiration in rats during hypothermia resulting from central drug administration.

The ability to induce a hypothermia resembling that of natural torpor would be greatly beneficial in medical and non-medical fields. At present, two procedures based on central nervous pharmacological manipulation have been shown to be effective in bringing core body temperature well below 30 °C in the rat, a non-hibernator: the first, based on the inhibition of a key relay in the central thermoregulatory pathway, the other, based on the activation of central adenosine A1 receptors. Although the role of mitochondria in the activation and maintenance of torpor has been extensively studied, no data are available for centrally induced hypothermia in non-hibernators. Thus, in the present work the respiration rate of mitochondria in the liver and in the kidney of rats following the aforementioned hypothermia-inducing treatments was studied. Moreover, to have an internal control, the same parameters were assessed in a well-consolidated model, i.e., mice during fasting-induced torpor. Our results show that state 3 respiration rate, which significantly decreased in the liver of mice, was unchanged in rats. An increase of state 4 respiration rate was observed in both species, although it was not statistically significant in rats under central adenosine stimulation. Also, a significant decrease of the respiratory control ratio was detected in both species. Finally, no effects were detected in kidney mitochondria in both species. Overall, in these hypothermic conditions liver mitochondria of rats remained active and apparently ready to be re-activated to produce energy and warm up the cells. These findings can be interpreted as encouraging in view of the finalization of a translational approach to humans.

Autonomic effects induced by pharmacological activation and inhibition of Raphe Pallidus neurons in anaesthetized adult pigs.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2 , whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174 bpm), systolic (from 87 to 170 mm Hg) and diastolic (from 51 to 98 mm Hg) arterial pressure, and end-tidal CO2 (from 49 to 62 mm Hg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

Phosphorylation and Dephosphorylation of Tau Protein During Synthetic Torpor.

Tau protein is of primary importance for many physiological processes in neurons, where it affects the dynamics of the microtubule system. When hyperphosphorylated (PP-Tau), Tau monomers detach from microtubules and tend to aggregate firstly in oligomers, and then in neurofibrillary tangles, as it occurs in a group of neurodegenerative disorders named thauopathies. A hypothermia-related accumulation of PP-Tau, which is quickly reversed after the return to normothermia, has been shown to occur in the brain of hibernators during torpor. Since, recently, in our lab, a hypothermic torpor-like condition (synthetic torpor, ST) was pharmacologically induced in the rat, a non-hibernator, the aim of the present work was to assess whether ST can lead to a reversible PP-Tau accumulation in the rat brain. PP-Tau was immunohistochemically assessed by staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau) in 19 brain structures, which were chosen mostly due to their involvement in the regulation of autonomic and cognitive functions in relation to behavioral states. During ST, AT8 staining was strongly expressed throughout the brain, while Tau-1 staining was reduced compared to control conditions. During the following recovery period, AT8 staining progressively reduced close to zero after 6 h from ST. However, Tau-1 staining remained low even after 38 h from ST. Thus, overall, these results show that ST induced an accumulation of PP-Tau that was, apparently, only partially reversed to normal during the recovery period. While the accumulation of PP-Tau may only depend on the physicochemical characteristics of the enzymes regulating Tau phosphorylation, the reverse process of dephosphorylation should be actively regulated, also in non-hibernators. In conclusion, in this work a reversible and widespread PP-Tau accumulation has been induced through a procedure that leads a non-hibernator to a degree of reversible hypothermia, which is comparable to that observed in hibernators. Therefore, the physiological mechanism involved in this process can sustain an adaptive neuronal response to extreme conditions, which may however lead to neurodegeneration when particular intensities and durations are exceeded.

c-Fos expression in the limbic thalamus following thermoregulatory and wake-sleep changes in the rat.

A cellular degeneration of two thalamic nuclei belonging to the "limbic thalamus", i.e., the anteroventral (AV) and mediodorsal (MD) nuclei, has been shown in patients suffering from Fatal Familial Insomnia (FFI), a lethal prion disease characterized by autonomic activation and severe insomnia. To better assess the physiological role of these nuclei in autonomic and sleep regulation, c-Fos expression was measured in rats during a prolonged exposure to low ambient temperature (Ta, - 10 °C) and in the first hours of the subsequent recovery period at normal laboratory Ta (25 °C). Under this protocol, the thermoregulatory and autonomic activation led to a tonic increase in waking and to a reciprocal depression in sleep occurrence, which was more evident for REM sleep. These effects were followed by a clear REM sleep rebound and by a rebound of Delta power during non-REM sleep in the following recovery period. In the anterior thalamic nuclei, c-Fos expression was (1) larger during the activity rather than the rest period in the baseline; (2) clamped at a level in-between the normal daily variation during cold exposure; (3) not significantly affected during the recovery period in comparison to the time-matched baseline. No significant changes were observed in either the MD or the paraventricular thalamic nucleus, which is also part of the limbic thalamus. The observed changes in the activity of the anterior thalamic nuclei appear, therefore, to be more specifically related to behavioral activation than to autonomic or sleep regulation.

Wake-sleep and cardiovascular regulatory changes in rats made obese by a high-fat diet.

Obesity is known to be associated with alterations in wake-sleep (WS) architecture and cardiovascular parameters. This study was aimed at assessing the possible influence of diet-induced obesity (DIO) on sleep homeostasis and on the WS state-dependent levels of arterial pressure (AP) and heart rate in the rat. Two groups of age-matched Sprague-Dawley rats were fed either a high-fat hypercaloric diet, leading to DIO, or a normocaloric standard diet (lean controls) for 8 weeks. While under general anesthesia, animals were implanted with instrumentation for the recording of electroencephalogram, electromyogram, arterial pressure, and deep brain temperature. The experimental protocol consisted of 48h of baseline, 12h of gentle handling, enhancing wake and depressing sleep, and 36-h post-handling recovery. Compared to lean controls, DIO rats showed: i) the same amount of rapid-eye movement (REM) and non-REM (NREM) sleep in the rest period, although the latter was characterized by more fragmented episodes; ii) an increase in both REM sleep and NREM sleep in the activity period; iii) a comparable post-handling sleep homeostatic response, in terms of either the degree of Delta power increase during NREM sleep or the quantitative compensation of the REM sleep loss at the end of the 36-h recovery period; iv) significantly higher levels of AP, irrespectively of the different WS states and of the changes in their intensity throughout the experimental protocol. Overall, these changes may be the reflection of a modification in the activity of the hypothalamic areas where WS, autonomic, and metabolic regulations are known to interact.

Wake-sleep, thermoregulatory, and autonomic effects of cholinergic activation of the lateral hypothalamus in the rat: a pilot study.

A major role in the wake-promoting effects of the activation of the lateral hypothalamus (LH) has been ascribed to a population of orexin (ORX)-containing neurons that send projections to central areas which regulate Wake-Sleep and autonomic function. Since, in the rat, a substantial amount of ORX neurons receive cholinergic projections from cells involved in Wake-Sleep regulation, the aim of this study was to assess the role played by LH cholinoceptive cells in Wake-Sleep and autonomic regulations. To this end, the effects of a microinjection of the cholinergic agonist Carbachol (CBL) into the LH were compared to those obtained through the activation of a wider cell population by the microinjection of the GABAA antagonist GABAzine (GBZ). The results of this pilot study showed that both drugs elicited the same behavioral and autonomic effects, those caused by GBZ being larger and longer-lasting than those following administration of CBL. Briefly, wakefulness was enhanced and sleep was depressed, and brain temperature and heart rate consistently increased, while mean arterial pressure showed only a mild increment. Surprisingly, the administration of the drug vehicle (SAL) elicited a similar pattern of Wake-Sleep effects which, although much smaller, were sufficient to mask any statistical significance between treatment and control data. In conclusion, the results of this work show that the arousal elicited by LH disinhibition by GABAzine is concomitant with autonomic responses set by the intervention of cold-defense mechanisms. Since the same response is elicited at a lower level by CBL administration, the hypothesis of an involvement of cholinoceptive ORX neurons in its generation is discussed.

Waking and sleeping in the rat made obese through a high-fat hypercaloric diet.

Sleep restriction leads to metabolism dysregulation and to weight gain, which is apparently the consequence of an excessive caloric intake. On the other hand, obesity is associated with excessive daytime sleepiness in humans and promotes sleep in different rodent models of obesity. Since no consistent data on the wake-sleep (WS) pattern in diet-induced obesity rats are available, in the present study the effects on the WS cycle of the prolonged delivery of a high-fat hypercaloric (HC) diet leading to obesity were studied in Sprague-Dawley rats. The main findings are that animals kept under a HC diet for either four or eight weeks showed an overall decrease of time spent in wakefulness (Wake) and a clear Wake fragmentation when compared to animals kept under a normocaloric diet. The development of obesity was also accompanied with the occurrence of a larger daily amount of REM sleep (REMS). However, the capacity of HC animals to respond to a "Continuous darkness" exposure condition (obtained by extending the Dark period of the Light-Dark cycle to the following Light period) with an increase of Sequential REMS was dampened. The results of the present study indicate that if, on one hand, sleep curtailment promotes an excess of energy accumulation; on the other hand an over-exceeding energy accumulation depresses Wake. Thus, processes underlying energy homeostasis possibly interact with those underlying WS behavior, in order to optimize energy storage.