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Obiettivo della tesi è stato quello di studiare il ruolo svolto dall’ipotalamo laterale (LH) nella regolazione dei processi di integrazione dell’attività autonomica e termoregolatoria con quella degli stati di veglia e sonno. A questo scopo, l’attività dell’LH è stata inibita per 6 ore (Esperimento A) mediante microiniezioni locali dell’agonista GABAA muscimolo nel ratto libero di muoversi, nel quale sono stati monitorati in continuo l’elelttroencefalogramma, l’elettromiogramma nucale, la pressione arteriosa (PA) e la temperatura ipotalamica (Thy) e cutanea. Gli animali sono stati studiati a temperatura ambientale (Ta) di 24°C e 10°C. I risultati hanno mostrato che l’inibizione acuta dell’LH riduce l’attività di veglia e sopprime la comparsa del sonno REM. Ciò avviene attraverso l’induzione di uno stato di sonno NREM caratterizzato da ipersincronizzazione corticale, con scomparsa degli stati transizionali al sonno REM. Quando l’animale è esposto a bassa Ta, tali alterazioni si associano a un ampio calo della Thy, che viene compensato da meccanismi vicarianti solo dopo un paio d’ore dall’iniezione. Sulla base di tali risultati, si è proceduto ad un ulteriore studio (Esperimento B) volto ad indagare il ruolo del neuropeptide ipocretina (prodotto in modo esclusivo a livello dell’LH) nei processi termoregolatori, mediante microiniezioni del medesimo nel bulbo rostrale ventromediale (RVMM), stazione cruciale della rete nervosa preposta all’attivazione dei processi termogenetici. La somministrazione di ipocretina è stata in grado di attivare la termogenesi e di potenziare la comparsa della veglia, con concomitante lieve incremento della PA e della frequenza cardiaca, quando effettuata alle Ta di 24°C o di 10°C, ma non alla Ta di 32°C. In conclusione, i risultati indicano che l’LH svolge un ruolo cruciale nella promozione degli stati di veglia e di sonno REM e, per tramite dell’ipocretina, interviene in modo coplesso a livello del RVMM nella regolazione dei processi di coordinamento dell'attività di veglia con quella termoregolatoria., Aim of my thesis was to investigate the lateral hypothalamus (LH) role in the regulation of autonomic and thermoregulatory integration processes with wakefulness and sleep states. For this purpose, LH was inhibited for up to 6 hours ( Experiment A) by local microinjections of the GABAA agonist muscimol in free behaving rats, implanted with electrodes for chronic electroencephalogram, nuchal electromyogram, arterial pressure (PA ), hypothalamic (Thy ) and skin temperature. The animals were exposed to 24 ° C and 10 ° C ambient temperature ( Ta ). The results showed that the acute inhibition of LH reduces wakefulness and suppresses the onset of REM sleep. This occurs through the induction of NREM sleep state, characterized by high cortical synchronization levels, with REM sleep transitional states disappearance. At low Ta, these results were associated with a large Thy decrease, reversed by compensatory systems a couple of hours after the injections. Starting from these results, we proceeded to investigate (Experiment B) the role of the hypocretin neuropeptide (produced exclusively in the LH ) in thermoregulatory processes, microinjecting hypocretin in the Rostral Ventromedial Medulla ( RVMM ), a crucial center of the neural network responsible for the thermogenetic processes activation. The hypocretin administration was able to activate thermogenesis and enhance wakefulness, with concomitant slight blood pressure and heart rate increase, at Ta of 24 ° C and 10 ° C, but not at Ta of 32 ° C. In conclusion, the results indicate that the LH plays a crucial role in wakefulness and REM sleep promotion and, through hypocretin, it intervenes in a complex way in regulating the wakefulness coordination processes, at the RVMM level.

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.

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.

Manual scoring of polysomnography data is labor-intensive and time-consuming, and most existing software does not account for subjective differences and user variability. Therefore, we evaluated a supervised machine learning algorithm, SomnivoreTM, for automated wake–sleep stage classification. We designed an algorithm that extracts features from various input channels, following a brief session of manual scoring, and provides automated wake-sleep stage classification for each recording. For algorithm validation, polysomnography data was obtained from independent laboratories, and include normal, cognitively-impaired, and alcohol-treated human subjects (total n = 52), narcoleptic mice and drug-treated rats (total n = 56), and pigeons (n = 5). Training and testing sets for validation were previously scored manually by 1–2 trained sleep technologists from each laboratory. F-measure was used to assess precision and sensitivity for statistical analysis of classifier output and human scorer agreement. The algorithm gave high concordance with manual visual scoring across all human data (wake 0.91 ± 0.01; N1 0.57 ± 0.01; N2 0.81 ± 0.01; N3 0.86 ± 0.01; REM 0.87 ± 0.01), which was comparable to manual inter-scorer agreement on all stages. Similarly, high concordance was observed across all rodent (wake 0.95 ± 0.01; NREM 0.94 ± 0.01; REM 0.91 ± 0.01) and pigeon (wake 0.96 ± 0.006; NREM 0.97 ± 0.01; REM 0.86 ± 0.02) data. Effects of classifier learning from single signal inputs, simple stage reclassification, automated removal of transition epochs, and training set size were also examined. In summary, we have developed a polysomnography analysis program for automated sleep-stage classification of data from diverse species. Somnivore enables flexible, accurate, and high-throughput analysis of experimental and clinical sleep studies., Frontiers in Neuroscience, 13, ISSN:1662-453X, ISSN:1662-4548

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.

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 48 h of baseline, 12 h 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.

Neural substrate of nausea is poorly understood, contrasting the wealth of knowledge about the emetic reflex. One of the reasons for this knowledge deficit is limited number and face validity of animal models of nausea. Our aim was to search for new physiological correlates of nausea in rats. Specifically, we addressed the question whether provocative motion (40-min rotation at 0.5 Hz) affects sleep architecture, brain temperature, heart rate (HR) and arterial pressure. Six adult male Sprague-Dawley rats were instrumented for recordings of EEG, nuchal electromyographic, hypothalamic temperature and arterial pressure. Provocative motion had the following effects: (1) total abolition of REM sleep during rotation and its substantial reduction during the first hour post-rotation (from 20 ± 3 to 5 ± 1.5 %); (2) reduction in NREM sleep, both during rotation (from 57 ± 6 to 19 ± 5 %) and during the first hour post-rotation (from 56 ± 3 to 41 ± 9 %); (3) fall in the brain temperature (from 37.1 ± 0.1 to 36.0 ± 0.1 °C); and (4) reduction in HR (from 375 ± 6 to 327 ± 7 bpm); arterial pressure was not affected. Ondansetron, a 5-HT3 antagonist, had no major effect on all observed parameters during both baseline and provocative motion. We conclude that in rats, provocative motion causes prolonged arousing effects, however without evidence of sympathetic activation that usually accompanies heightened arousal. Motion-induced fall in the brain temperature complements and extends our previous observations in rats and suggests that similar to humans, provocative motion triggers coordinated thermoregulatory response, leading to hypothermia in this species.