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La possibilità di indurre stati ipotermici ed ipometabolici come il torpore o l’ibernazione in animali non ibernanti può avere dei risvolti utili nella pratica medica, in quanto permetterebbe di trarre vantaggio dagli effetti benefici dell’ipotermia senza gli effetti compensatori negativi causati dalla risposta omeostatica dell’organismo. Con questo lavoro vogliamo proporre un nuovo approccio, che coinvolge il blocco farmacologico dell’attività dei neuroni nel bulbo rostroventromediale (RVMM), un nucleo troncoencefalico che si è rivelato essere uno snodo chiave nella regolazione della termogenesi attraverso il controllo dell’attività del tessuto adiposo bruno, della vasomozione cutanea e del cuore. Nel nostro esperimento, sei iniezioni consecutive del agonista GABAA muscimolo nel RVMM, inducono uno stato reversibile di profonda ipotermia (21°C al Nadir) in ratti esposti ad una temperatura ambientale di 15°C. Lo stato ipotermico/ipomentabolico prodotto dall’inibizione dei neuroni del RVMM mostra forti similitudini col torpore naturale, anche per quanto concerne le modificazioni elettroencefalografiche osservate durante e dopo la procedura. Come negli ibernati naturali, nei ratti cui viene inibito il controllo della termogenesi si osserva uno spostamento verso le regioni lente delle spettro di tutte le frequenze dello spettro EEG durante l’ipotermia, ed un forte incremento dello spettro EEG dopo il ritorno alla normotermia, in particolare della banda Delta (0,5-4Hz) durante il sonno NREM. Per concludere, questi risultati dimostrano che l’inibizione farmacologica selettiva di un nucleo troncoencefalico chiave nel controllo della termogenesi è sufficiente per indurre uno stato di psuedo-torpore nel ratto, una specie che non presenta stati di torpore spontaneo. Un approccio di questo tipo può aprire nuove prospettive per l’utilizzo in ambito medico dell’ipotermia., The possibility to mimic hypomethabolic and hypothermic states like torpor or hibernation in non-hibernating animals may be useful in medical practice since the beneficial effects of deep hypothermia could be obtained without the concomitant elicitation of adverse autonomic compensatory responses. Until now, tentatives of induction of such states have been focused on the inhibition of cellular metabolism at systemic level but worked only in animals already able to enter torpor. Here we show a new approach, that involves the pharmacological blockade of the activity of the neurons within the Rostral Ventromedia Medulla (RVMM), a brainstem area that has been shown to be a key thermogenetic relay that controls the brown adipose tissue, the cutaneous blood vessels and the heart. In our experiment, six consecutive injections, aimed to block the thermogenetic drive by the activation of the GABAA receptors in RVMM neurons, induced a reversible deep hypothermia (21°C at nadir) state in free behaving rats exposed to 15°C ambient temperature. The hypothermic/hypometabolic state produced by prolonged inhibition of RVMM neurons strongly resembles the naturally occurring torpor, even in the EEG changes observed during and after the procedure. Like animals that spontaneously show hibernation or torpor, rats with the thermogentic drive blocked show a left-shift of all the EEG frequencies during the hypothermia, and a strong increase in EEG power spectrum after the return to thermoneutrality, in particular in the EEG Delta band (0.5-4.0 Hz) during NREM sleep. In conclusion, these results show that the selective pharmacological inhibition of a key brainstem area of the cold-defence pathway is sufficient in inducing a topor-like state in the rat, a species that does not enter torpor spontaneously. Such an approach may open new perspectives in the use of deep hypothermia in several medical conditions.

Objectives: The Rostral Ventromedial Medulla (RVMM) is a key area in the control of autonomic function and metabolism, promoting thermogenesis (1). In the present study, the effects on sleep and cardiovascular parameters of the induction of a torpor-like state by the pharmacological inhibition of RVMM were investigated in the rat. Methods: Male Sprague–Dawley rats (n = 18, 300–350 g) adapted to an ambient temperature (Ta) of 25C, were implanted, under general anaesthesia (diazepam, 5 mg/kg, i.m., ketamine, 100 mg/kg, i.p.), with electrodes for EEG and EMG recording, a thermistor for detecting hypothalamic temperature (Thy), a catheter for arterial blood pressure recording, and a microcannula within the RVMM. After a week of recovery, animals were kept for 3 days at Ta 15C and then they were microinjected within the RVMM (1 injection/h, for 6 h) with either the GABAA agonist muscimol [1 mM, 100 nl; Group 1 (n = 6) and Group 2 (n = 6)] or saline [0.9%, 100 nl, Group 3 (n = 6)]. In order to favour the return to normothermia, 1 h after the last injection Ta was raised to 28C for Groups 1 and 3 and, for Group 2, to 37C, which was kept for 1 h and then taken to 28C. Results: Muscimol induced a deep hypothermia (Thy: 22.8 ± 0.8C) which was accompanied by: (i) a reduction of EEG activity; (ii) a decrease in heart rate (HR), from 440 ± 13 to 201 ± 12 bpm; (iii) a substantial maintenance of mean arterial pressure (MAP) at normal levels. Group 2 recovered normothermia faster than Group 1. Recovery was characterized by: (i) a progressive normalization of HR; (ii) the occurrence of a peak in MAP compared to saline (Group 3, 92 ± 4 mmHg), that was larger (P < 0.01) in Group 2 (125 ± 4 mmHg) than in Group 1 (116 ± 2 mmHg); (iii) the building up, in Groups 1 and 2, of a large SWS rebound in NREM sleep and of a not significant rebound in REM sleep. Conclusion: The effects of RVMM inhibition on cardiovascular and sleep parameters resembled those observed in natural torpor (2, 3). The increase in MAP observed during the recovery may be favoured by an excess in sympathetic activity. The effects on sleep observed after the recovery of normothermia indicate that the hypothermic bout and/or the rewarming generated a homeostatic need for SWS, but not for REM sleep. References: 1) Morrison S, Nakamura K. Front Biosci (2011) 16: 74–104. 2) Swoap SJ, Gutilla MJ. Am J Physiol RICP (2009) 297:R769–774. 3) Deboer T, Tobler I. Sleep (2003) 26: 567–72.

La possibilità di indurre stati ipotermici ed ipometabolici come il torpore o l’ibernazione in animali non ibernanti può avere dei risvolti utili nella pratica medica, in quanto permetterebbe di trarre vantaggio dagli effetti benefici dell’ipotermia senza gli effetti compensatori negativi causati dalla risposta omeostatica dell’organismo. Con questo lavoro vogliamo proporre un nuovo approccio, che coinvolge il blocco farmacologico dell’attività dei neuroni nel bulbo rostroventromediale (RVMM), un nucleo troncoencefalico che si è rivelato essere uno snodo chiave nella regolazione della termogenesi attraverso il controllo dell’attività del tessuto adiposo bruno, della vasomozione cutanea e del cuore. Nel nostro esperimento, sei iniezioni consecutive del agonista GABAA muscimolo nel RVMM, inducono uno stato reversibile di profonda ipotermia (21°C al Nadir) in ratti esposti ad una temperatura ambientale di 15°C. Lo stato ipotermico/ipomentabolico prodotto dall’inibizione dei neuroni del RVMM mostra forti similitudini col torpore naturale, anche per quanto concerne le modificazioni elettroencefalografiche osservate durante e dopo la procedura. Come negli ibernati naturali, nei ratti cui viene inibito il controllo della termogenesi si osserva uno spostamento verso le regioni lente delle spettro di tutte le frequenze dello spettro EEG durante l’ipotermia, ed un forte incremento dello spettro EEG dopo il ritorno alla normotermia, in particolare della banda Delta (0,5-4Hz) durante il sonno NREM. Per concludere, questi risultati dimostrano che l’inibizione farmacologica selettiva di un nucleo troncoencefalico chiave nel controllo della termogenesi è sufficiente per indurre uno stato di psuedo-torpore nel ratto, una specie che non presenta stati di torpore spontaneo. Un approccio di questo tipo può aprire nuove prospettive per l’utilizzo in ambito medico dell’ipotermia., The possibility to mimic hypomethabolic and hypothermic states like torpor or hibernation in non-hibernating animals may be useful in medical practice since the beneficial effects of deep hypothermia could be obtained without the concomitant elicitation of adverse autonomic compensatory responses. Until now, tentatives of induction of such states have been focused on the inhibition of cellular metabolism at systemic level but worked only in animals already able to enter torpor. Here we show a new approach, that involves the pharmacological blockade of the activity of the neurons within the Rostral Ventromedia Medulla (RVMM), a brainstem area that has been shown to be a key thermogenetic relay that controls the brown adipose tissue, the cutaneous blood vessels and the heart. In our experiment, six consecutive injections, aimed to block the thermogenetic drive by the activation of the GABAA receptors in RVMM neurons, induced a reversible deep hypothermia (21°C at nadir) state in free behaving rats exposed to 15°C ambient temperature. The hypothermic/hypometabolic state produced by prolonged inhibition of RVMM neurons strongly resembles the naturally occurring torpor, even in the EEG changes observed during and after the procedure. Like animals that spontaneously show hibernation or torpor, rats with the thermogentic drive blocked show a left-shift of all the EEG frequencies during the hypothermia, and a strong increase in EEG power spectrum after the return to thermoneutrality, in particular in the EEG Delta band (0.5-4.0 Hz) during NREM sleep. In conclusion, these results show that the selective pharmacological inhibition of a key brainstem area of the cold-defence pathway is sufficient in inducing a topor-like state in the rat, a species that does not enter torpor spontaneously. Such an approach may open new perspectives in the use of deep hypothermia in several medical conditions.

Introduction: The periods of torpor are typically followed by the occurrence of high-Delta power NREM sleep (NREMS) (1). In the present study, this phenomenon was investigated in rats through the induction of a torpor like state by the pharmacological inhibition of the Rostral Ventromedial Medulla (RVMM), a key area in the central nervous control of metabolism, promoting thermogenesis (2). Methods: Eighteen male Sprague-Dawley rats (300–350 g) were surgically implanted, under general anaesthesia (Diazepam, 5 mg/ kg, i.m., ketamine, 100 mg/kg, i.p.), with electrodes for chronic EEG recording, a thermistor for the detection of the hypothalamic temperature (Thy), a catheter for arterial blood presssure recording, and a microcannula for drug delivery within the RVMM. Before the experiment, animals were exposed for three days to ambient conditions that are known to favor the occurrence of a natural torpor bout: ambient temperature (Ta) 15C, constant darkness and a highfat diet. Three groups of animals were microinjected within the RVMM (1 injection/h, for 6 h) with the GABAA agonist muscimol (1 mM, 100 nl; Group 1 (n = 6) and Group 2 (n = 6)) or with saline (0.9%, 100 nl, group 3 (n = 6)). One hour after the last injections, Ta was raised to 28C for groups 1 and 3, and to 37C for Group 2 to favour the return to normothermia. Results: In both Group 1 and 2, a deep hypothermia was observed during the 6-h microinjection period (Thy fell to 22.80 ± 0.8C) which was accompanied by a decrease of EEG activity and a progressive shift of the EEG spectral power towards the low-frequency region. After the injection period, EEG activity showed a progressive increase and the EEG spectral power shifted to the high frequency region. In particular, after the recovery of normothermia, a powerful intensification of Delta power in NREMS was observed, which lasted about 6 h. The peak in Delta power was significantly higher in Group 1 than in Group 2 (P < 0.01). No significant effects were observed in Group 3. Conclusion: The dynamics of EEG changes during and after the torpor-like state induction were similar to those described in natural torpor. The increase in Delta power in NREMS was attenuated under environmental conditions promoting the restoration of normothermia, suggesting a role for the degree of sympathetic activation during restoration in its determination. 1. Deboer T, Tobler I. Sleep. 2003; 26: 567–72. 2. Morrison S, Nakamura K. Front Biosci., 2011; 16: 74–104.

Introduction: The antagonism of hypocretins (HCRT) has been proposed as a new approach to the treatment of insomnia. Since HCRT is exclusively synthesized by neurons located within the lateral hypothalamus (LH) [1], in the present study, we have evaluated the effects on sleep induced by a prolonged pharmacological inhibition of the LH at both normal laboratory (25C) and low (10C) ambient temperature (Ta), a condition which is known to dysfacilitate sleep ocurrence [2]. Methods: Twelve male Sprague-Dawley rats (300–350 g) were surgically implanted, under general anaesthesia (Diazepam, 5mg/ kg, i.m., ketamine, 100 mg/kg, i.p.), with electrodes for chronic EEG recording, a thermistor for the detection of the hypothalamic temperature, a catheter for arterial blood presssure recording and two microcannulas for drug delivery within the LH. Two groups of animals were studied under a 6-day (D1–D6) protocol. Animals from group 1 (n = 5) were injected with either the GABAA agonist Muscimol (100 nl, 1 mM, 1 injection/h for 6 h, from 11:00 h to 16:00 h) or vehicle (saline, 0.9%; according to the former protocol) during D2 and D5, respecitvely. In group 2 (n = 7), the same experimental protocol was carried out, but Ta was lowered to 10C in both D2 and D5, from 9:00 h to 17:00 h. Results: In the six experimental hours, muscimol injection induced a significant increase in NREM sleep amount at both Ta 25C (+6099 ± 574 s) and Ta 10C (+4896 ± 408 s) compared to what observed after vehicle (P < 0.01, for both comparisons). In both groups, a concomitant suppression of REM sleep was obsereved after muscimol injection Conclusion: The acute inhibition of LH neurons in free behaving rats produces a long bout of NREM sleep, even in environmental condition unfavourable to sleep occurence. This effect can be explained by the inhibition of the activity of HCRT neurons, which would favor the activation of the circuits promoting NREM sleep occurrence. The suppression of REM sleep may be due to the concomitant inhibithion of a group of melanin concentrating hormone (MCH) neurons located in the LH, which have been shown to be involved in REM sleep regulation [3]. 1. Sakurai T, Mieda M, Tsujino N. Ann N Y Acad Sci 2010, 1200: 149–61. 2. Cerri, M. Ocampo-Garces, A. Amici R, Baracchi F, Capitani P, Jones CA, Luppi M, Perez E, Parmeggiani PL, and Zamboni G. Sleep 2005, 28: 694:705. 3. Sapin E, Berod A, Leger L, Herman PA, Luppi PH, Peyron C. PLoS One 2010, 5: e11766.

Studies conducted on anaesthetized animals have shown that neurons within the rostral ventromedial medulla (RVMM) can modulate the cardiac sympathetic outflow. Disinhibition of RVMM neurons by microinjection of the GABA-A antagonist bicuculline methiodide induces a strong increase in heart rate (HR), but inhibition of the same neurons by microinjection of the GABA-A agonist muscimol does not produce clear effects on HR. However, general anaesthesia affects sympathetic outflow and does not permit a evaluation of heart rate variability (HRV) measure. In the present study the role of RVMM neurons in mediating tonic cardiac sympathetic outflow has been assessed in free behaving rats. HRV has been evaluated in six free behaving rats before and after microinjection of muscimol (1mM) within the RVMM. HRV was analyzed within both the time domain (R-R mean; R-R standard deviation (STD-RR); root mean square successive difference (RMSSD)) and frequency domain (High (HF, 0.6–2.4 Hz) and Low Frequency (LF, 0.06–0.6 Hz) bands). Effects of injections were separated into six temporal blocks according to the dynamics of changes in core temperature following RVMM inhibition. At the end of the experiment, animals the site of injection was histologically verified. The power of the LF band, expression of cardiovascular sympathetic drive, was clearly reduced following RVMM neurons inhibition, suggesting that RVMM neurons participate in maintaining tonic cardiac sympathetic outflow.

Recent research proposes that putative sympathetic premotor neurons controlling cutaneous vasomotion are located within the rostral ventromedial medulla (RVMM), an area delimited rostrally by the nucleus of the facial nerve (RVMM(fn)) and caudally by the rostral pole of the inferior olive (RVMM(io)). Data from several experiments have shown that cutaneous blood flow is increased by the inhibition and reduced by the activation of neurons within the RVMM(fn), but no data are available on the role of the RVMM(io) on cutaneous vasomotion. To clarify the role of RVMM(fn) and RVMM(io) neurons in controlling cutaneous vasomotion, microinjection of the GABAA agonist muscimol and GABAA antagonist bicuculline methiodide were performed within the RVMM(fn) or the RVMM(io). A massive peripheral vasodilatation was evoked by muscimol injection within both the two areas, while disinhibition of either RVMM(fn) or RVMM(io) neurons produced, respectively, a reversal of the increase in cutaneous blood flow induced by warm exposure and an unexpected rapid cutaneous vasodilatation. The results show that two intermingled sets of neurons are located within the RVMM: a vasoconstrictory and thermoregulatory set, which is tonically active, is located in both the RVMM(io) and the RVMM(fn), while a vasodilatatory and non-thermoregulatory set, which is tonically inhibited, is located in the RVMM(io).