Marco Mastrotto

The Inhibition of Neurons in the Central Nervous Pathways for Thermoregulatory Cold Defense Induces a Suspended Animation State in the Rat

The possibility of inducing a suspended animation state similar to natural torpor would be greatly beneficial in medical science, since it would avoid the adverse consequence of the powerful autonomic activation evoked by external cooling. Previous attempts to systemically inhibit metabolism were successful in mice, but practically ineffective in nonhibernators. Here we show that the selective pharmacological inhibition of key neurons in the central pathways for thermoregulatory cold defense is sufficient to induce a suspended animation state, resembling natural torpor, in a nonhibernator. In rats kept at an ambient temperature of 15°C and under continuous darkness, the prolonged inhibition (6 h) of the rostral ventromedial medulla, a key area of the central nervous pathways for thermoregulatory cold defense, by means of repeated microinjections (100 nl) of the GABAA agonist muscimol (1 mm), induced the following: (1) a massive cutaneous vasodilation; (2) drastic drops in deep brain temperature (reaching a nadir of 22.44 ± 0.74°C), heart rate (from 440 ± 13 to 207 ± 12 bpm), and electroencephalography (EEG) power; (3) a modest decrease in mean arterial pressure; and (4) a progressive shift of the EEG power spectrum toward slow frequencies. After the hypothermic bout, all animals showed a massive increase in NREM sleep Delta power, similarly to that occurring in natural torpor. No behavioral abnormalities were observed in the days following the treatment. Our results strengthen the potential role of the CNS in the induction of hibernation/torpor, since CNS-driven changes in organ physiology have been shown to be sufficient to induce and maintain a suspended animation state.

Neuronal mechanism for acute mechanosensitivity in tactile-foraging waterfowl

Significance Like vision, audition, and olfaction, mechanosensation is a fundamental way in which animals interact with the environment, but it remains the least well understood at the cellular and molecular levels. Here, we explored evolutionary changes that contribute to the enhancement of mechanosensitivity in tactile-foraging ducks. We found that the somatosensory neurons that innervate the duck bill can detect physical force much more efficiently than analogous cells in other species, such as mice. Furthermore, ducks exhibit an increase in the number of neurons dedicated to this task in their sensory ganglia and a decrease in the number of neurons that detect temperature. Our findings provide an explanation for the acute mechanosensitivity of the duck bill at the level of somatosensory neurons. Relying almost exclusively on their acute sense of touch, tactile-foraging birds can feed in murky water, but the cellular mechanism is unknown. Mechanical stimuli activate specialized cutaneous end organs in the bill, innervated by trigeminal afferents. We report that trigeminal ganglia (TG) of domestic and wild tactile-foraging ducks exhibit numerical expansion of large-diameter mechanoreceptive neurons expressing the mechano-gated ion channel Piezo2. These features are not found in visually foraging birds. Moreover, in the duck, the expansion of mechanoreceptors occurs at the expense of thermosensors. Direct mechanical stimulation of duck TG neurons evokes high-amplitude depolarizing current with a low threshold of activation, high signal amplification gain, and slow kinetics of inactivation. Together, these factors contribute to efficient conversion of light mechanical stimuli into neuronal excitation. Our results reveal an evolutionary strategy to hone tactile perception in vertebrates at the level of primary afferents.

Neuronal UCP1 expression suggests a mechanism for local thermogenesis during hibernation

Significance Mammalian hibernators can reduce their metabolic rate by 95% and body temperature to 2 °C. However, their central and peripheral nervous systems retain activity even in cold, through unknown mechanisms. We report here that neurons from hibernating squirrels express uncoupling protein 1 (UCP1), a protein known as a heat generator in brown adipose tissue. We show that squirrel UCP1 acts as the typical thermogenic protein and is up-regulated during torpor, suggesting its thermogenic capability is important during hibernation. Accordingly, we found that the temperature of squirrel brain during the deep torpor associated with hibernation is warmer than the surrounding tissues. We hypothesize that neuronal UCP1 allows squirrels to withstand the long hibernation season and tolerate temperatures prohibitively low for survival and neuronal function in nonhibernating species. Hibernating mammals possess a unique ability to reduce their body temperature to ambient levels, which can be as low as −2.9 °C, by active down-regulation of metabolism. Despite such a depressed physiologic phenotype, hibernators still maintain activity in their nervous systems, as evidenced by their continued sensitivity to auditory, tactile, and thermal stimulation. The molecular mechanisms that underlie this adaptation remain unknown. We report, using differential transcriptomics alongside immunohistologic and biochemical analyses, that neurons from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) express mitochondrial uncoupling protein 1 (UCP1). The expression changes seasonally, with higher expression during hibernation compared with the summer active state. Functional and pharmacologic analyses show that squirrel UCP1 acts as the typical thermogenic protein in vitro. Accordingly, we found that mitochondria isolated from torpid squirrel brain show a high level of palmitate-induced uncoupling. Furthermore, torpid squirrels during the hibernation season keep their brain temperature significantly elevated above ambient temperature and that of the rest of the body, including brown adipose tissue. Together, our findings suggest that UCP1 contributes to local thermogenesis in the squirrel brain, and thus supports nervous tissue function at low body temperature during hibernation.

The direct cooling of the preoptic-hypothalamic area elicits the release of thyroid stimulating hormone during wakefulness but not during REM sleep.

Thermoregulatory responses to temperature changes are not operant during REM sleep (REMS), but fully operant in non-REM sleep and wakefulness. The specificity of the relationship between REMS and the impairment of thermoregulation was tested by eliciting the reflex release of Thyrotropin Releasing Hormone (TRH), which is integrated at hypothalamic level. By inducing the sequential secretion of Thyroid Stimulating Hormone (TSH) and Thyroid Hormone, TRH intervenes in the regulation of obligatory and non-shivering thermogenesis. Experiments were performed on male albino rats implanted with epidural electrodes for EEG recording and 2 silver-copper wire thermodes, bilaterally placed in the preoptic-hypothalamic area (POA) and connected to small thermoelectric heat pumps driven by a low-voltage high current DC power supply. In preliminary experiments, a thermistor was added in order to measure hypothalamic temperature. The activation of TRH hypophysiotropic neurons by the thermode cooling of POA was indirectly assessed, in conditions in which thermoregulation was either fully operant (wakefulness) or not operant (REMS), by a radioimmunoassay determination of plasmatic levels of TSH. Different POA cooling were performed for 120 s or 40 s at current intensities of 80 mA and 125 mA, respectively. At both current intensities, POA cooling elicited, with respect to control values (no cooling current), a significant increase in plasmatic TSH levels in wakefulness, but not during REMS. These results confirm the inactivation of POA thermal sensitivity during REMS and show, for the first time, that this inactivation concerns also the fundamental endocrine control of non-shivering thermogenesis.

Enhanced slow-wave EEG activity and thermoregulatory impairment following the inhibition of the lateral hypothalamus in the rat.

Neurons within the lateral hypothalamus (LH) are thought to be able to evoke behavioural responses that are coordinated with an adequate level of autonomic activity. Recently, the acute pharmacological inhibition of LH has been shown to depress wakefulness and promote NREM sleep, while suppressing REM sleep. These effects have been suggested to be the consequence of the inhibition of specific neuronal populations within the LH, i.e. the orexin and the MCH neurons, respectively. However, the interpretation of these results is limited by the lack of quantitative analysis of the electroencephalographic (EEG) activity that is critical for the assessment of NREM sleep quality and the presence of aborted NREM-to-REM sleep transitions. Furthermore, the lack of evaluation of the autonomic and thermoregulatory effects of the treatment does not exclude the possibility that the wake-sleep changes are merely the consequence of the autonomic, in particular thermoregulatory, changes that may follow the inhibition of LH neurons. In the present study, the EEG and autonomic/thermoregulatory effects of a prolonged LH inhibition provoked by the repeated local delivery of the GABAA agonist muscimol were studied in rats kept at thermoneutral (24°C) and at a low (10°C) ambient temperature (Ta), a condition which is known to depress sleep occurrence. Here we show that: 1) at both Tas, LH inhibition promoted a peculiar and sustained bout of NREM sleep characterized by an enhancement of slow-wave activity with no NREM-to-REM sleep transitions; 2) LH inhibition caused a marked transitory decrease in brain temperature at Ta 10°C, but not at Ta 24°C, suggesting that sleep changes induced by LH inhibition at thermoneutrality are not caused by a thermoregulatory impairment. These changes are far different from those observed after the short-term selective inhibition of either orexin or MCH neurons, suggesting that other LH neurons are involved in sleep-wake modulation.

Molecular basis of tactile specialization in the duck bill

Significance Tactile-specialist birds of the Anatidae family possess unique mechanosensory abilities with which they efficiently select edible matter in muddy water without visual or olfactory cues. Mechanical stimuli are transmitted by trigeminal mechanoreceptors innervating the bill, a highly specialized tactile organ. We show mechanosensory specialization in ducks involves the formation of functional rapidly adapting mechanoreceptors prior to hatching. Unlike in visually foraging chicken, most trigeminal neurons in ducks are touch receptors, which develop following a unique pattern of neurotrophic factor receptor expression and produce robust mechano-current via the Piezo2 channel with novel properties. Our results uncover possible evolutionary adaptations contributing to potentiation of mechanoreception in an organ-specific manner and reveal the molecular identity of a neuronal mechanotransducer with prolonged inactivation kinetics. Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Somatosensory Neurons Enter a State of Altered Excitability during Hibernation

Hibernation in mammals involves prolonged periods of inactivity, hypothermia, hypometabolism, and decreased somatosensation. Peripheral somatosensory neurons play an essential role in the detection and transmission of sensory information to CNS and in the generation of adaptive responses. During hibernation, when body temperature drops to as low as 2°C, animals dramatically reduce their sensitivity to physical cues [1, 2]. It is well established that, in non-hibernators, cold exposure suppresses energy production, leading to dissipation of the ionic and electrical gradients across the plasma membrane and, in the case of neurons, inhibiting the generation of action potentials [3]. Conceivably, such cold-induced elimination of electrogenesis could be part of a general mechanism that inhibits sensory abilities in hibernators. However, when hibernators become active, the bodily functions-including the ability to sense environmental cues-return to normal within hours, suggesting the existence of mechanisms supporting basal functionality of cells during torpor and rapid restoration of activity upon arousal. We tested this by comparing properties of somatosensory neurons from active and torpid thirteen-lined ground squirrels (Ictidomys tridecemlineatus). We found that torpid neurons can compensate for cold-induced functional deficits, resulting in unaltered resting potential, input resistance, and rheobase. Torpid neurons can generate action potentials but manifest markedly altered firing patterns, partially due to decreased activity of voltage-gated sodium channels. Our results provide insights into the mechanism that preserves somatosensory neurons in a semi-active state, enabling fast restoration of sensory function upon arousal. These findings contribute to the development of strategies enabling therapeutic hypothermia and hypometabolism.

Sensing Force by Trigeminal Neurons of Acutely Mechanosensitive Birds

Mechanosensation is a fundamental way animals interact with the environment, but it remains the least well understood at cellular and molecular level. Somatosensory ganglia of the standard laboratory species house a highly diverse population of neurons, where low-threshold mechanoreceptors - the neurons that innervate light touch receptors in the skin - represent only a small fraction. This heterogeneity significantly impedes progress in understanding functional roles of somatosensory neurons in light touch perception. Here, we explored functional specialization of somatosensory ganglia from animals which have taken the sense of touch to the extreme - tactile foraging ducks. These animals have acutely mechanosensitive bill innervated by trigeminal (TG) neurons, and as such provide an opportunity to study general principles of mechanotransduction from an unconventional standpoint. We found that, in contrast to species without tactile specialization, the majority (85%) of duck TG neurons are large-diameter myelinated mechanoreceptors expressing the mechano-gated ion channel Piezo2. Electrophysiological analyses showed that mechanosensitivity of duck TG neurons has been optimized in three ways. Compared to mouse cells, duck neurons exhibit (i) lowered threshold of mechano-activation, (ii) elevated signal amplification gain, and (iii) prolonged kinetics of inactivation, all of which increase the amount of depolarizing charge entering the cell upon mechanical stimulation. Thus, duck TG neurons have augmented intrinsic ability to convert mechanical force into excitatory ionic current, which explains the acute mechanosensory properties of the duck bill. Our studies emphasize a key role of the intrinsic mechanosensory ability of somatosensory neurons in touch physiology, reveal an evolutionary strategy utilized by vertebrates to hone tactile perception, and suggest a novel model system to study the sense of touch at the cellular and molecular level.Schneider ER, Gracheva EO, Bagriantsev SN et al, PNAS 2014 (e-pub Sept 22).