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  1. 1. 2984 • The Journal of Neuroscience, February 13, 2013 • 33(7):2984 –2993Behavioral/CognitiveThe Inhibition of Neurons in the Central Nervous Pathwaysfor Thermoregulatory Cold Defense Induces a SuspendedAnimation State in the RatMatteo Cerri, Marco Mastrotto,* Domenico Tupone,* Davide Martelli, Marco Luppi, Emanuele Perez,Giovanni Zamboni, and Roberto AmiciDepartment of Biomedical and NeuroMotor Sciences, Alma Mater Studiorum–University of Bologna, 40126 Bologna ItalyThe possibility of inducing a suspended animation state similar to natural torpor would be greatly beneficial in medical science, since itwould avoid the adverse consequence of the powerful autonomic activation evoked by external cooling. Previous attempts to systemicallyinhibit metabolism were successful in mice, but practically ineffective in nonhibernators. Here we show that the selective pharmacolog-ical inhibition of key neurons in the central pathways for thermoregulatory cold defense is sufficient to induce a suspended animationstate, resembling natural torpor, in a nonhibernator. In rats kept at an ambient temperature of 15°C and under continuous darkness, theprolonged inhibition (6 h) of the rostral ventromedial medulla, a key area of the central nervous pathways for thermoregulatory colddefense, by means of repeated microinjections (100 nl) of the GABAA agonist muscimol (1 mM), induced the following: (1) a massivecutaneous vasodilation; (2) drastic drops in deep brain temperature (reaching a nadir of 22.44 Ϯ 0.74°C), heart rate (from 440 Ϯ 13 to207 Ϯ 12 bpm), and electroencephalography (EEG) power; (3) a modest decrease in mean arterial pressure; and (4) a progressive shift ofthe EEG power spectrum toward slow frequencies. After the hypothermic bout, all animals showed a massive increase in NREM sleepDelta 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 physiologyhave been shown to be sufficient to induce and maintain a suspended animation state.Introduction esized that two basic players take part in inducing a reduction inSuspended animation is a temporary and fully reversible condi- metabolism: humoral factors (Andrews, 2007) and the CNStion characterized by hypometabolism and deep hypothermia, (Drew et al., 2007).during which physiological functions are slowed down. In mam- The intervention of humoral factors has recently been high-mals, this condition spontaneously occurs under the form of tor- lighted by the induction of a suspended animation state in mice,por and hibernation, which are triggered by environmental a species where torpor occurs naturally, by the administration offactors (Melvin and Andrews, 2009). The cellular and molecular several substances interfering with cell metabolism (Scanlan etmechanisms of natural suspended animation are still unknown al., 2004; Blackstone et al., 2005; Gluck et al., 2006; Zhang et al.,(Carey et al., 2003), but since hypothermia is preceded by a met- 2006). However, the translational outcomes of this approachabolic rate reduction (Heldmaier et al., 2004), it has been hypoth- (Lee, 2008) have been hampered, so far, by the failure to replicate these results in nonhibernators (Haouzi et al., 2008, Zhang et al., 2009). Although the intervention of the CNS in determining nat-Received July 27, 2012; revised Dec. 19, 2012; accepted Dec. 20, 2012. ural suspended animation remains largely unexplored (Drew et Author contributions: M.C., G.Z., and R.A. designed research; M.C., M.M., D.T., and D.M. performed research; M.C., al., 2007), the increase in heat loss and the decrease in heat gen-M.M., D.T., and M.L. analyzed data; M.C., E.P., G.Z., and R.A. wrote the paper. eration that follow the chemical manipulation of the central ner- This work is supported by the Ministero dell’Universita e della Ricerca Scientifica (MIUR), Italy, (PRIN 2008, Project ` vous pathways for thermoregulatory cold defense (Morrison and2008FY7K9S). *D.T. and M.M. equally contributed to this work. Nakamura, 2011) suggests that the reduction in metabolism may The authors declare no competing financial interests. also be actively driven by the CNS. Correspondence should be addressed to Matteo Cerri, Department of Biomedical and NeuroMotor Sci- A key area in the central nervous pathways for thermoregula-ences, Alma Mater Studiorum–University of Bologna Piazza di Porta S. Donato 2, 40126 Bologna, Italy. tory cold defense is the rostral ventromedial medulla (RVMM), aE-mail: M. Mastrotto’s present address: Department of Biological and Biomedical Sciences, Yale University, New Haven, region including the raphe pallidus (RPa) and the raphe magnus,CT 06520. where the putative sympathetic premotor neurons to the brown D. Tupone’s present address: Department of Neurological Surgery, Oregon Health and Science University, Port- adipose tissue (BAT), the cutaneous blood vessel, and the heartland, OR 97239-3098. are located (Cano et al., 2003). The activation of RPa neurons has D. Martelli’s present address: Systems Neurophysiology Division, Florey Institute of Neuroscience and MentalHealth, University of Melbourne, Parkville, Victoria 3010, Australia. been shown to promote nonshivering (Morrison et al., 1999) and DOI:10.1523/JNEUROSCI.3596-12.2013 shivering (Nakamura and Morrison, 2011) thermogenesis, cuta-Copyright © 2013 the authors 0270-6474/13/332984-10$15.00/0 neous vasoconstriction (Blessing and Nalivaiko, 2001), and an
  2. 2. Waking and Sleeping following Water Deprivation in theRatDavide Martelli1,2, Marco Luppi1, Matteo Cerri1, Domenico Tupone1,3, Emanuele Perez1,Giovanni Zamboni1*, Roberto Amici11 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Bologna, Italy, 2 Systems Neurophysiology Division, Florey NeuroscienceInstitutes, University of Melbourne, Melbourne, Australia, 3 Oregon National Primate Research Center, Oregon Health & Science University, Portland, Oregon, United Statesof America Abstract Wake-sleep (W-S) states are affected by thermoregulation. In particular, REM sleep (REMS) is reduced in homeotherms under a thermal load, due to an impairment of hypothalamic regulation of body temperature. The aim of this work was to assess whether osmoregulation, which is regulated at a hypothalamic level, but, unlike thermoregulation, is maintained across the different W-S states, could influence W-S occurrence. Sprague-Dawley rats, kept at an ambient temperature of 24uC and under a 12 h:12 h light-dark cycle, were exposed to a prolonged osmotic challenge of three days of water deprivation (WD) and two days of recovery in which free access to water was restored. Two sets of parameters were determined in order to assess: i) the maintenance of osmotic homeostasis (water and food consumption; changes in body weight and fluid composition); ii) the effects of the osmotic challenge on behavioral states (hypothalamic temperature (Thy), motor activity, and W-S states). The first set of parameters changed in WD as expected and control levels were restored on the second day of recovery, with the exception of urinary Ca++ that almost disappeared in WD, and increased to a high level in recovery. As far as the second set is concerned, WD was characterized by the maintenance of the daily oscillation of Thy and by a decrease in activity during the dark periods. Changes in W-S states were small and mainly confined to the dark period: i) REMS slightly decreased at the end of WD and increased in recovery; ii) non-REM sleep (NREMS) increased in both WD and recovery, but EEG delta power, a sign of NREMS intensity, decreased in WD and increased in recovery. Our data suggest that osmoregulation interferes with the regulation of W-S states to a much lesser extent than thermoregulation. Citation: Martelli D, Luppi M, Cerri M, Tupone D, Perez E, et al. (2012) Waking and Sleeping following Water Deprivation in the Rat. PLoS ONE 7(9): e46116. doi:10.1371/journal.pone.0046116 Editor: Gianluca Tosini, Morehouse School of Medicine, United States of America Received January 18, 2012; Accepted August 28, 2012; Published September 24, 2012 Copyright: ß 2012 Martelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been funded by Research Funds for Fundamental and Applied Research (RFO Funds) from the University of Bologna (http://www.eng. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: giovanni.zamboni@unibo.itIntroduction hormone arginine vasopressin (AVP) was kept at the same levels in the different wake-sleep (W-S) states, indicates that hypothalamic Physiological regulation is known to be different during rapid osmoregulation is not impaired during REMS [7]. This suggestseye movement sleep (REMS) when compared to non rapid eye that the change in hypothalamic integrative activity in this sleepmovement sleep (NREMS) [1]. In particular, during NREMS a stage should concern structures related to thermoregulation, ratherstable autonomic outflow is observed in the presence of a fully than the whole hypothalamus as previously hypothesized [1].operant homeostatic control of physiological variables [1–4]. In Since clarification regarding the issue of specificity of thecontrast, during REMS a high variability of the autonomic impairment in physiological regulation during REMS may beoutflow, which leads to large irregularities in arterial blood relevant for the understanding of this sleep state, we sought to testpressure, heart rate, and respiratory rhythm, is concomitant with the observed independence of osmoregulation from autonomican impairment of thermoregulation [1–4]. This impairment has changes in sleep. To this end, REMS occurrence and overall W-Sbeen considered to be a visible consequence of a change in regulation were assessed in rats during exposure to a three-dayhypothalamic integrative activity, disabling, during REMS, the water deprivation (WD) protocol. This condition is known toautonomic feedbacks sustaining body homeostasis [1]. According- constitute an osmotic challenge leading to the progressively, Wake-Sleep (W-S) states change when a tonic maintenance of engagement of the whole set of mechanisms maintaining bodythe hypothalamic regulation is needed, as it occurs during the fluid homeostasis [8,9]. Moreover, since a REMS rebound wasexposure to a low ambient temperature (Ta). In these conditions, observed during the recovery (R) period following cold exposureWake increases, NREMS is variably affected and REMS is always [5,6,10,11,12,13], W-S assessment was continued for two daysreduced or even suppressed in proportion to the Ta levels after water was once again made freely available. Basically, two[2,4,5,6]. sets of parameters were assessed: i) those concerning the However, the recent finding from our laboratory that, following maintenance of osmotic homeostasis (water and food consump-a central osmotic stimulation, the release of the antidiureticPLOS ONE | 1 September 2012 | Volume 7 | Issue 9 | e46116
  3. 3. Environ Sci Pollut ResDOI 10.1007/s11356-012-1266-5 ECOTOXICOLOGY AND ENVIRONMENTAL TOXICOLOGY : NEW CONCEPTS, NEW TOOLSEffects of chronic exposure to radiofrequencyelectromagnetic fields on energy balance in developing ratsAmandine Pelletier & Stéphane Delanaud &Pauline Décima & Gyorgy Thuroczy & René de Seze &Matteo Cerri & Véronique Bach & Jean-Pierre Libert &Nathalie LoosReceived: 4 July 2012 / Accepted: 16 October 2012# Springer-Verlag Berlin Heidelberg 2012Abstract The effects of radiofrequency electromagnetic peripheral vasoconstriction, which was confirmed in an ex-fields (RF-EMF) on the control of body energy balance in periment with the vasodilatator prazosin. Exposure to RF-developing organisms have not been studied, despite the EMF also increased daytime food intake (+0.22 gh−1). Mostinvolvement of energy status in vital physiological functions. of the observed effects of RF-EMF exposure were dependentWe examined the effects of chronic RF-EMF exposure on Ta. Exposure to RF-EMF appears to modify the functioning(900 MHz, 1 Vm−1) on the main functions involved in body of vasomotor tone by acting peripherally through α-energy homeostasis (feeding behaviour, sleep and thermoreg- adrenoceptors. The elicited vasoconstriction may restrict bodyulatory processes). Thirteen juvenile male Wistar rats were cooling, whereas energy intake increases. Our results showexposed to continuous RF-EMF for 5 weeks at 24 °C of air that RF-EMF exposure can induce energy-saving processestemperature (Ta) and compared with 11 non-exposed animals. without strongly disturbing the overall sleep pattern.Hence, at the beginning of the 6th week of exposure, thefunctions were recorded at Ta of 24 °C and then at 31 °C. Keywords Radiofrequency electromagnetic field . Sleep .We showed that the frequency of rapid eye movement sleep Feeding behaviour . Thermoregulation . Young ratepisodes was greater in the RF-EMF-exposed group, indepen-dently of Ta (+42.1 % at 24 °C and +31.6 % at 31 °C). Theother effects of RF-EMF exposure on several sleep parameters Introductionwere dependent on Ta. At 31 °C, RF-EMF-exposed animalshad a significantly lower subcutaneous tail temperature Body energy balance is a relevant factor in growing organisms,(−1.21 °C) than controls at all sleep stages; this suggested since energy is required for vital functions, thermoregulation and tissue synthesis (in that order). The regulation of energyResponsible editor: Philippe Garrigues balance involves a complicated interaction between energyA. Pelletier : S. Delanaud : P. Décima : V. Bach : J.-P. Libert : intake (feeding behaviour), energy saving (sleep), energy-N. Loos (*) dissipating mechanisms (vasomotricity) and energy produc-PériTox Laboratory (EA 4285-UMI01), Faculty of Medicine, tion, all of which are controlled by hypothalamic structuresJules Verne University of Picardy (UPJV), (Blessing 2003; El Hajjaji et al. 2011; Himms-Hagen 1995).3 rue des Louvels, CS 13602,80036 Amiens cedex 1, France Several studies have shown that these functions are ine-mail: competition; sleep and feeding cannot be performed simul- taneously, for example. In the rat, there is a positive corre-G. Thuroczy : R. de Seze lation between meal size and the durations of both non-rapidPériTOX Laboratory (EA 4285-UMI01), VIVA/TOXI,National Institute of Industrial Environment and Risks (INERIS), eye movement sleep (NREMS) and rapid eye movementParc ALATA BP2, sleep (REMS): the larger the energy intake, the longer the60550 Verneuil-en-Halatte, France rat will sleep (Danguir and Nicolaidis 1985). As far as thermal status and feeding behaviour are concerned, bodyM. CerriDepartment of Human and General Physiology, cooling and body heating stimulate and reduce food intake,Alma Mater Studiorum-University of Bologna, respectively (De Vries et al. 1993; El Hajjaji et al. 2011).Bologna, Italy Thus, an animal in a cold environment consumes extra food
  4. 4. J. Sleep Res. (2010) 19, 394–399 Osmoregulation and sleepdoi: 10.1111/j.1365-2869.2009.00810.xHypothalamic osmoregulation is maintained across thewake–sleep cycle in the ratMARCO LUPPI1, DAVIDE MARTELLI1, ROBERTO AMICI1, FRANCESCABARACCHI2, MATTEO CERRI1, DANIELA DENTICO1, EMANUELEP E R E Z 1 and G I O V A N N I Z A M B O N I 11 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Bologna, Italy and 2Department of HumanPhysiology, University of Milan, Milan, ItalyAccepted in revised form 12 October 2009; received 17 June 2009 SUMMARY In different species, rapid eye movement sleep (REMS) is characterized by a thermoregulatory impairment. It has been postulated that this impairment depends on a general insufficiency in the hypothalamic integration of autonomic function. This study aims to test this hypothesis by assessing the hypothalamic regulation of body fluid osmolality during the different wake–sleep states in the rat. Arginine-vasopressin (AVP) plasma levels were determined following intracerebroventricular (ICV) infusions of artificial cerebrospinal fluid (aCSF), either isotonic or made hypertonic by the addition of NaCl at three different concentrations (125, 250 and 500 mm). Animals were implanted with a cannula within a lateral cerebral ventricle for ICV infusions and with electrodes for the recording of the electroencephalogram. ICV infusions were made in different animals during Wake, REMS or non-REM sleep (NREMS). The results show that ICV infusion of hypertonic aCSF during REMS induced an increase in AVP plasma levels that was not different from that observed during either Wake or NREMS. These results suggest that the thermoregulatory impairment that characterizes REMS does not depend on a general impairment in the hypothalamic control of body homeostasis. k e y w o r d s arginine-vasopressin, body homeostasis, hypothalamus, osmoregulation, rapid eye movement sleep, wake–sleep cycle The issue of generalizing the impairment of thermoregula-INTRODUCTION tion during REMS to all hypothalamic integrative mechanismsRapid eye movement sleep (REMS) is characterized by has not been addressed so far from an experimental point ofchanges in physiological regulation, which differentiate this view. A way to address this problem is that of studyingsleep state from Wake and non-rapid eye movement sleep regulations that have the highest degree of integration at the(NREMS; Parmeggiani, 2005). In particular, thermoregulation hypothalamic level and that therefore display a more intensehas been shown to be impaired during REMS in different disruption in case of a local impairment of integrative activity.species (Heller, 2005; Parmeggiani, 2003), and this change has In line with this view, the aim of this work was to study thebeen interpreted as the outcome of a more general impairment regulation of body fluid osmolality during the different wake–in the hypothalamic integration of autonomic function, which sleep (W–S) states, as it represents a function that is almostwould explain the high degree of instability of the autonomic completely integrated at the hypothalamic level in mammalsactivity during REMS (Parmeggiani, 1980, 1988, 1994). (Denton et al., 1996). In these species, the most efficient defense of osmolality isCorrespondence: Giovanni Zamboni, MD, Department of Human andGeneral Physiology, Alma Mater Studiorum-University of Bologna, provided by the release of arginine-vasopressin (AVP), thePiazza P.ta S. Donato, 2, I-40126 Bologna, Italy. Tel.: +39 051 anti-diuretic hormone, by hypothalamic magnocellular neu-2091742; fax: +39 051 2091737; e-mail: rons of the supraoptic nucleus (SON) and paraventricular394 Ó 2010 European Sleep Research Society
  5. 5. Neuroscience 165 (2010) 984 –995CUTANEOUS VASODILATION ELICITED BY DISINHIBITION OF THECAUDAL PORTION OF THE ROSTRAL VENTROMEDIAL MEDULLA OFTHE FREE-BEHAVING RATM. CERRI,* G. ZAMBONI, D. TUPONE, D. DENTICO, tissue (BAT) thermogenesis (Morrison, 2001; Cano et al.,M. LUPPI, D. MARTELLI, E. PEREZ AND R. AMICI 2003; Nakamura et al., 2005) and cardiac rate (Cao andDipartimento di Fisiologia Umana e Generale, Alma Mater Studiorum Morrison, 2003). Antagonism of GABAA receptors in theUniversità di Bologna, Piazza di Porta S. Donato 2, 40126, Italy RVMM produces an increase in body temperature in the anaesthetized animal through both a peripheral vasocon-Abstract—Putative sympathetic premotor neurons control- striction (Blessing and Nalivaiko, 2001) and an increase inling cutaneous vasomotion are contained within the rostral BAT thermogenesis (Morrison et al., 1999). Conversely,ventromedial medulla (RVMM) between levels corresponding, the injection into the same area of a GABAA agonist leadsrostrally, to the rostral portion of the nucleus of the facial to vasodilation of the tail skin in the anaesthetized ratnerve (RVMM(fn)) and, caudally, to the rostral pole of the (Blessing and Nalivaiko, 2001), and to hypothermia (Za-inferior olive (RVMM(io)). Cutaneous vasoconstrictor premo- retsky et al., 2003) and tail vasodilation (Vianna et al.,tor neurons in the RVMM(fn) play a major role in mediatingthermoregulatory changes in cutaneous vasomotion that reg- 2008) in conscious rats.ulate heat loss. To determine the role of neurons in the Studies employing injections of the transneuronal ret-RVMM(io) in regulating cutaneous blood flow, we examined rograde tracer, pseudorabies virus within the rat tail arterythe changes in the tail and paw skin temperature of free- wall, have localized putative sympathetic premotor neu-behaving rats following chemically-evoked changes in the rons controlling cutaneous blood vessels throughout theactivity of neurons in the RVMM(io). Microinjection of the RVMM, between the levels corresponding to the rostralGABAA agonist, muscimol, within either the RVMM(fn) or portion of nucleus of the facial nerve and the rostral portionthe RVMM(io) induced a massive peripheral vasodilation; mi-croinjection of the GABAA antagonist bicuculline methiodide of the inferior olive (Smith et al., 1998; Nakamura et al.,within the RVMM(fn) reversed the increase in cutaneous 2004; Toth et al., 2006). Interestingly, a relative differ-blood flow induced by warm exposure and, unexpectedly, ence in neuronal phenotype has been suggested betweendisinhibition of RVMM(io) neurons produced a rapid cutane- the rostral portion of the RVMM, containing more putativeous vasodilation. We conclude that the tonically-active neu- glutamatergic neurons expressing the vesicular glutamaterons driving cutaneous vasoconstriction, likely sympathetic transporter, VGLUT3, and the caudal portion of the RVMM,premotor neurons previously described in the RVMM(fn), arealso located in the RVMM(io). However, in the RVMM(io), where more serotoninergic neurons have been identifiedthese are accompanied by a population of neurons that re- (Nakamura et al., 2004; Stornetta et al., 2005; Toth et al.,ceives a tonically-active GABAergic inhibition in the con- 2006). From both anatomical and physiological evidence, itscious animal and that promotes a cutaneous vasodilation has been proposed that the neural substrate in the RVMMupon relief of this inhibition. Whether the vasodilator neurons for the control of cutaneous vasomotion is represented bylocated in the RVMM(io) play a role in thermoregulation re- a set of VGLUT3-positive, glutamatergic neurons directlymains to be determined. © 2010 IBRO. Published by Elsevier projecting to the intermediolateral column (IML) of theLtd. All rights reserved. spinal cord (Nakamura et al., 2004). These data have ledKey words: infrared thermography, thermoregulation, sym- to a theoretical paradigm in which sympathetic outflow topathetic nervous system, cutaneous vasomotion, muscimol, cutaneous blood vessels is proportional to the level ofbicuculline methiodide. activity of the RVMM glutamatergic sympathetic premotor neurons (Nakamura et al., 2004). The evaluation of the physiological role of RVMM neu-The rostral ventromedial medulla (RVMM) contains popu- rons controlling cutaneous blood flow has been until nowlations of sympathetic premotor neurons controlling sev- limited to the rostral portion of the RVMM, within the rostro-eral autonomic functions, including cutaneous vasomotion caudal level of the nucleus of the facial nerve (RVMM(fn))(Smith et al., 1998; Nagashima et al., 2000; Blessing and (Tanaka et al., 2002; Ootsuka and Blessing, 2006), whereNalivaiko, 2001; Nakamura et al., 2004), brown adipose the more VGLUT3 positive neurons are located. No data*Corresponding author. Tel: ϩ39-051-2091756; fax: ϩ39-051-2091737. are available on the role of the more caudal portion ofE-mail address: (M. Cerri).Abbreviations: AP, arterial pressure; BAT, brown adipose tissue; EKG, RVMM neurons, within the rostro-caudal section of theelectrocardiogram; HR, heart rate; IML, intermediolateral nucleus of rostral pole of the inferior olive (RVMM(io)), in controllingthe spinal cord; RVMM, rostral ventromedial medulla; RVMM(fn), ros- cutaneous vasomotion.tral ventromedial medulla at the level of the facial nucleus; RVMM(io), The aim of the present study is a more extensiverostral ventromedial medulla at the level of the rostral inferior olivarynucleus; Ta, ambient temperature; Thy, hypothalamic temperature; characterization of the RVMM physiological role in control-Tpaw, paw temperature; Ttail, tail temperature. ling cutaneous vasomotion, with special focus on the cau-0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2009.10.068 984
  6. 6. European Journal of NeuroscienceEuropean Journal of Neuroscience, Vol. 30, pp. 651–661, 2009 doi:10.1111/j.1460-9568.2009.06848.x BEHAVIORAL NEUROSCIENCEc-Fos expression in preoptic nuclei as a marker of sleeprebound in the ratDaniela Dentico,1 Roberto Amici,1 Francesca Baracchi,1,2 Matteo Cerri,1 Elide Del Sindaco,1 Marco Luppi,1Davide Martelli,1 Emanuele Perez1 and Giovanni Zamboni11 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Piazza P.ta S. Donato, 2, I-40126Bologna, Italy2 Research Division, Department of Anesthesiology, University of Michigan, Ann Arbor, MI, USAKeywords: c-Fos, cold exposure, median preoptic nucleus, P-CREB, ventrolateral preoptic nucleusAbstractThermoregulation is known to interfere with sleep, possibly due to a functional interaction at the level of the preoptic area (POA).Exposure to low ambient temperature (Ta) induces sleep deprivation, which is followed by sleep rebound after a return to laboratoryTa. As two POA subregions, the ventrolateral preoptic nucleus (VLPO) and the median preoptic nucleus (MnPO), have beenproposed to have a role in sleep-related processes, the expression of c-Fos and the phosphorylated form of the cAMP ⁄ Ca2+-responsive element-binding protein (P-CREB) was investigated in these nuclei during prolonged exposure to a Ta of )10 °C and inthe early phase of the recovery period. Moreover, the dynamics of the sleep rebound during recovery were studied in a separategroup of animals. The results show that c-Fos expression increased in both the VLPO and the MnPO during cold exposure, but not ina specific subregion within the VLPO cluster counting grid (VLPO T-cluster). During the recovery, concomitantly with a large rapid eyemovement sleep (REMS) rebound and an increase in delta power during non-rapid eye movement sleep (NREMS), c-Fos expressionwas high in both the VLPO and the MnPO and, specifically, in the VLPO T-cluster. In both nuclei, P-CREB expression showedspontaneous variations in basal conditions. During cold exposure, an increase in expression was observed in the MnPO, but not inthe VLPO, and a decrease was observed in both nuclei during recovery. Dissociation in the changes observed between c-Fosexpression and P-CREB levels, which were apparently subject to state-related non-regulatory modulation, suggests that the sleep-related changes observed in c-Fos expression do not depend on a P-CREB-mediated pathway.IntroductionThe preoptic area (POA) is known to be a key structure in the Exposure to low ambient temperature (Ta) represents a useful toolregulation of body temperature (Romanovsky, 2007), and has been for inducing physiological sleep deprivation, which is followed by aclearly recognized as a sleep-promoting site (Szymusiak et al., 2007), sleep rebound after a return to normal laboratory conditionsprobably representing the diencephalic substrate of the integration (Parmeggiani, 2003). The thermoregulatory impairment that charac-between thermoregulation and sleep-related processes (Parmeggiani, terizes rapid eye movement sleep (REMS) makes cold exposure2003). A relevant feature of the POA is that almost 25% of neurons particularly challenging for REMS (Parmeggiani, 2003; Heller, 2005).therein increase their activity at sleep onset and ⁄ or during sleep In particular, during prolonged exposure to very low Tas, REMSoccurrence (Szymusiak et al., 2007). It has been proposed that two pressure was shown to increase dramatically, leading to an intensePOA subregions, the ventrolateral preoptic nucleus (VLPO) and the REMS rebound during the following recovery (Amici et al., 1994,median preoptic nucleus (MnPO), may have a key role in sleep-related 1998, 2008; Cerri et al., 2005). Extreme cold is apparently lessprocesses on the basis of the results of unit recording (Szymusiak challenging for non-rapid eye movement sleep (NREMS), as, under aet al., 1998; Suntsova et al., 2002, 2007), anatomical tracer (Sherin 24-h exposure to a Ta of )10 °C protocol, the changes in both NREMSet al., 1996, 1998; Steininger et al., 2001; Uschakov et al., 2007) and amount and the power density in the delta band of the electroenceph-immunohistochemical studies (analysis of c-Fos expression) (Morgan alogram were smaller than those observed in REMS amount, during& Curran, 1991; Sherin et al., 1996; Gong et al., 2000, 2004). Sleep both the exposure and the following recovery (Cerri et al., 2005).deprivation studies have suggested that, in both nuclei, c-Fos In the present study, in order to better assess the role of the VLPOactivation is related to sleep occurrence and ⁄ or to an increase in and MnPO in sleep-related processes under a long-term physiologicalsleep pressure (Gong et al., 2004; Gvilia et al., 2006a,b). sleep deprivation protocol, c-Fos expression was studied during prolonged exposure to a Ta of )10 °C and the subsequent early recovery at laboratory Ta. In addition, as prolonged exposure to a Ta ofCorrespondence: Dr Giovanni Zamboni, as above. )10 °C was shown to dampen the maximum accumulation capacity ofE-mail: the second messenger cAMP at the POA level (Zamboni et al., 1996,Received 4 December 2008, revised 11 June 2009, accepted 16 June 2009 1999, 2004), the expression of the phosphorylated form of theª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
  7. 7. J. Sleep Res. (2008) 17, 166–179 Sleep in animalsdoi: 10.1111/j.1365-2869.2008.00658.xCold exposure impairs dark-pulse capacity to induce REM sleepin the albino ratFRANCESCA BARACCHI1,2, GIOVANNI ZAMBONI1, MATTEO CERRI1,ELIDE DEL SINDACO1, DANIELA DENTICO1, CHRISTINE ANN JONES1,M A R C O L U P P I 1 , E M A N U E L E P E R E Z 1 and R O B E R T O A M I C I 11 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Italy and 2Department of Anesthesiology,Research Division, University of Michigan, Ann Arbor, USAAccepted in revised form 24 February 2008; received 30 November 2007 SUMMARY In the albino rat, a REM sleep (REMS) onset can be induced with a high probability and a short latency when the light is suddenly turned off (dark pulse, DP) during non- REM sleep (NREMS). The aim of this study was to investigate to what extent DP delivery could overcome the integrative thermoregulatory mechanisms that depress REMS occurrence during exposure to low ambient temperature (Ta). To this aim, the efficiency of a non-rhythmical repetitive DP (3 min each) delivery during the first 6-h light period of a 12 h : 12 h light–dark cycle in inducing REMS was studied in the rat, through the analysis of electroencephalogram, electrocardiogram, hypothalamic temperature and motor activity at different Tas. The results showed that DP delivery triggers a transition from NREMS to REMS comparable to that which occurs spontaneously. However, the efficiency of DP delivery in inducing REMS was reduced during cold exposure to an extent comparable with that observed in spontaneous REMS occurrence. Such impairment was associated with low Delta activity and high sympathetic tone when DPs were delivered. Repetitive DP administration increased REMS amount during the delivery period and a subsequent negative REMS rebound was observed. In conclusion, DP delivery did not overcome the integrative thermo- regulatory mechanisms that depress REMS in the cold. These results underline the crucial physiological meaning of the mutual exclusion of thermoregulatory activation and REMS occurrence, and support the hypothesis that the suspension of the central control of body temperature is a prerequisite for REMS occurrence. keywords dark pulse, low ambient temperature, non-REM sleep to REM sleep transition, preotpic-anterior hypothalamus, REM sleep, REM sleep homeostasis change in physiological regulation that shifts from a homeo-INTRODUCTION static to a non-homeostatic modality during REMS (Par-The wake–sleep cycle consists of the alternation of three meggiani, 2005). Such a shift is more relevant for a regulation,different states, Wake, non-REM sleep (NREMS) and REM such as thermoregulation, that needs a high degree ofsleep (REMS), which are usually identified on the basis of the integration and largely depends on the activity of regulatorylevel of brain cortical and muscle activity. From a physiolog- structures of the hypothalamus (Parmeggiani, 2003). Thus,ical point of view, the major feature that differentiates REMS REMS occurrence needs to be finely regulated, since itfrom both Wake and NREMS consists of an operational represents a physiological challenge for the organism, partic- ularly when under unfavourable ambient conditions, such asCorrespondence: Roberto Amici, Department of Human and GeneralPhysiology, Alma Mater Studiorum-University of Bologna, Piazza during the exposure to a low ambient temperature (Ta).P.ta S. Donato, 2 I-40126 Bologna, Italy. Tel.: +39-051-2091735; fax: In different species, the passage from NREMS to REMS has+39-051-2091737; e-mail: been shown not to occur abruptly (Gottesmann, 1996;166 Ó 2008 European Sleep Research Society
  8. 8. REM SleepCold Exposure and Sleep in the Rat: REM Sleep Homeostasis and Body SizeRoberto Amici, MD1; Matteo Cerri, MD, PhD1; Adrian Ocampo-Garcés, MD, PhD2; Francesca Baracchi, PhD1,3; Daniela Dentico, MD, PhD1;Christine Ann Jones, PhD1; Marco Luppi, PhD1; Emanuele Perez, MD1; Pier Luigi Parmeggiani, MD1; Giovanni Zamboni, MD11 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Italy; 2Programa de Fisiología y Biofísica,Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile; 3Department of Anaesthesiology- ResearchDivision, University of Michigan, Ann Arbor, MI Study Objectives: Exposure to low ambient temperature (Ta) depress- beyond a “fast rebound” threshold corresponding to 22% of the daily es REM sleep (REMS) occurrence. In this study, both short and long- REMS need. A slow REMS rebound apparently allowed the animals term homeostatic aspects of REMS regulation were analyzed during to fully restore the previous REMS loss during the following 3 days of cold exposure and during subsequent recovery at Ta 24°C. recovery. Design: EEG activity, hypothalamic temperature, and motor activ- Conclusion: Comparing the present data on rats with data from ear- ity were studied during a 24-h exposure to Tas ranging from 10°C to lier studies on cats and humans, it appears that small mammals have –10°C and for 4 days during recovery. less tolerance for REMS loss than large ones. In small mammals, this Setting: Laboratory of Physiological Regulation during the Wake-Sleep low tolerance may be responsible on a short-term basis for the shorter Cycle, Department of Human and General Physiology, Alma Mater wake-sleep cycle, and on long-term basis, for the higher percentage of Studiorum-University of Bologna. REMS that is quickly recovered following REMS deprivation. Subjects: 24 male albino rats. Keywords: REM sleep, low ambient temperature, REM sleep homeo- Interventions: Animals were implanted with electrodes for EEG re- stasis, REM sleep rebound, body size, theta power density. cording and a thermistor to measure hypothalamic temperature. Citation: Amici R; Cerri M; Ocampo-Garcés A; Baracchi F; Dentico D; Measurements and Results: REMS occurrence decreased propor- Jones CA; Luppi M; Perez E; Parmeggiani PL; Zamboni G. Cold ex- tionally with cold exposure, but a fast compensatory REMS rebound posure and sleep in the rat: rem sleep homeostasis and body size. occurred during the first day of recovery when the previous loss went SLEEP 2008;31(5):708-715.MANY STUDIES HAVE SHOWN THAT A SLEEP DEFICIT In spite of this, many different “short-term” (minutes/hours)INDUCES A SUBSEQUENT INCREASE IN THE DURA- or “long-term” (hours/days) sleep deprivation studies seekingTION AND/OR IN THE INTENSITY OF SLEEP AND THAT a homeostatically regulated sleep parameter have shown thatthe occurrence of sleep reduces sleep propensity. The outcome NREMS is substantially regulated in terms of intensity, andof the regulation of such a balance between sleep and wake has REMS in terms of duration.2 However, some contradictory as-been addressed as “sleep homeostasis”.1,2 pects arising from “extended” (days/weeks) sleep deprivation The major hindrance in a quantitative approach to sleep studies and/or from the comparison of animal and human stud-homeostasis lies in the fact that sleep consists of two different ies still leave this topic open to discussion.2,8-11states, NREM sleep (NREMS) and REM sleep (REMS), which As far as NREMS is concerned, the power density in thecyclically alternate on an ultradian basis. In particular: (1) it is delta band (approximately, 0.5-4.5 Hz) of the electroencepha-not possible to carry out a selective NREMS deprivation with- logram (EEG), not NREMS duration, is considered to be theout interfering with REMS occurrence; (2) selective REMS homeostatically regulated parameter in NREMS and is com-deprivation procedures have been shown to affect to some ex- monly taken as an index of NREMS intensity.1,2,12 Such a regu-tent the quality of NREMS during deprivation3,4; (3) a complex lation appears to be disrupted following an extended period ofinteraction between NREMS and REMS rebounds has been either sleep deprivation or sleep restriction.9,13observed following different sleep deprivation protocols5,6; and REMS appears to be precisely regulated in terms of its dura-(4) NREMS and REMS regulation are influenced differently by tion on both a short-term and long-term basis. The short-termcircadian rhythmicity.7 component is expressed as a function of the ultradian wake-sleep cycle. Within a species, the duration of the interval between two consecutive REM episodes (REMS interval) appears to be di-Disclosure Statement rectly related to the duration of the preceding REMS episode,This was not an industry supported study. Dr. Cerri has received research but not to the duration of the subsequent REMS episode.14-17support from the European Sleep Research Society through a sponsor- The long-term component of REMS regulation is expressed inship from Sanofi-Aventis. The other authors have indicated no financial the total amount of REMS during the days following a totalconflicts of interest. sleep or selective REM sleep deprivation. A precise REMS con- servation has been observed in both the cat and the rat, sinceSubmitted for publication July, 2007 the rebound in REMS has been found to be proportional to theAccepted for publication December, 2007Address correspondence to: Roberto Amici, MD, Department of Human total loss of REMS during the deprivation period. 11,18-22 Thisand General Physiology, Alma Mater Studiorum-University of Bologna, precise quantitative regulation of total REMS amount may notPiazza P.ta S. Donato, 2, I-40126 Bologna, Italy; Tel: +39-051-2091735; occur following an extended period of sleep deprivation orFax: +39-051-2091737; E-mail: sleep restriction.9,13,23 Whereas changes in EEG power are anSLEEP, Vol. 31, No. 5, 2008 708 REM Sleep Homeostasis and Body Size—Amici et al
  9. 9. Available online at Behavioural Brain Research 187 (2008) 254–261 Research report Lithium affects REM sleep occurrence, autonomic activity and brain second messengers in the ratଝ Christine Ann Jones ∗ , Emanuele Perez, Roberto Amici, Marco Luppi, Francesca Baracchi, Matteo Cerri, Daniela Dentico, Giovanni Zamboni Dipartimento di Fisiologia umana e generale, Universit` di Bologna, Bologna, Italy a Received 5 July 2006; received in revised form 7 August 2007; accepted 17 September 2007 Available online 20 September 2007Abstract The effects of a single intraperitoneal administration of lithium, a drug used to prevent the recurrence of mania in bipolar disorders, weredetermined in the rat by studying changes in: (i) the wake–sleep cycle; (ii) autonomic parameters (hypothalamic and tail temperature, heart rate);(iii) the capacity to accumulate cAMP and IP3 in the preoptic-anterior hypothalamic region (PO-AH) and in the cerebral cortex (CC) under anhypoxic stimulation at normal laboratory and at low ambient temperature (Ta ). In the immediate hours following the injection, lithium induced:(i) a significant reduction in REM sleep; (ii) a non-significant reduction in the delta power density of the EEG in NREM sleep; (iii) a significantdecrease in the concentration of cAMP in PO-AH at normal laboratory Ta ; (iv) a significant increase of IP3 concentration in CC following exposureto low Ta . The earliest and most sensitive effects of lithium appear to be those concerning sleep. These changes are concomitant with biochemicaleffects that, in spite of a systemic administration of the substance, may be differentiated according to the second messenger involved, the brainregion and the ambient condition.© 2007 Elsevier B.V. All rights reserved.Keywords: cAMP; IP3 ; REM sleep; Autonomic activity; Preoptic anterior-hypothalamic area; Cerebral cortex1. Introduction This study has been originated from two sets of separate experimental results following REM sleep deprivation induced Lithium is traditionally used in bipolar disorder to prevent by exposure to a very low ambient temperature (Ta ) for 48 h.the recurrence of episodes of mania [6] and also appears to The first set of results showed that early recovery was character-be very effective in the management of acute manic episodes ized by a rebound of REM sleep occurring with a longer delay[26,27,35]. A common symptom of these mood disorders is and a lower rate than that observed for deprivations induced byinsomnia [8], but a consistent alteration of sleep appears to affect shorter exposures, that is, characterized by a smaller REM sleepthe first REM episode, which occurs at a latency shorter than loss [3]. The second set showed that these changes in REM sleepthat observed in normal individuals [7]. Although a shorten- recovery were concomitant with changes in the brain accumula-ing of REM sleep latency is seen in other psychopathological tion capacity of second messenger: (i) the capacity to accumulatedisturbances [38], it has been observed that the normalization adenosine 3 :5 cyclic monophosphate (cAMP) was significantlyof REM sleep occurrence is concomitant with a symptomatic reduced in the preoptic-anterior hypothalamic area (PO-AH), theimprovement in depressed patients [8]. Likewise, the adminis- thermoregulatory region involved in the control of the negativetration of lithium to manic depressive patients increases REM interaction between sleep and homeothermia, but not in the cere-sleep latency [11]. bral cortex (CC), whose bioelectrical activity is used to classify sleep stages; (ii) the capacity to accumulate 1,4,5-trisphosphate (IP3 ) significantly increased in both PO-AH and CC [48]. ଝ Supported by a grant from MIUR, Italy. ∗ Since several observations show that cAMP and IP3 brain Corresponding author at: Dipartimento di Fisiologia umana e generale,Piazza di Porta San Donato 2, 40126 Bologna, BO, Italy. Tel.: +39 051 2091726; levels are influenced by lithium [15,22,34], we sought to studyfax: +39 051 2091737. whether the similarity of changes in REM sleep occurrence E-mail address: (C.A. Jones). caused by exposure to low Ta and by the administration of the0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbr.2007.09.017
  10. 10. Neuroscience 140 (2006) 711–721CORTICOTROPIN RELEASING FACTOR INCREASES IN BROWNADIPOSE TISSUE THERMOGENESIS AND HEART RATE THROUGHDORSOMEDIAL HYPOTHALAMUS AND MEDULLARYRAPHE PALLIDUSM. CERRIa,b AND S. F. MORRISONa* the raphe pallidus. © 2006 Published by Elsevier Ltd ona Neurological Sciences Institute, Oregon Health & Science University, behalf of IBRO.505 Northwest 185th Avenue, Beaverton, OR 97006, USAb Key words: sympathetic nerve activity, energy balance, Dipartimento di Fisiologia Umana e Generale Universita’ degli Studi di stress, hyperthermia.Bologna Piazza di Porta S. Donato, 40126, Bologna, ItalyAbstract—Corticotropin releasing factor, acting at hypotha- Corticotropin releasing factor (CRF), identified in 1981lamic corticotropin releasing factor receptors, contributes to (Vale et al., 1981), is a neuropeptide hormone synthesizedthe neural signaling pathways mediating stress-related re- by neurons in the parvocellular paraventricular nucleussponses, as well as those involved in maintaining energy (PVN) of the hypothalamus that acts on pituitary cells tobalance homeostasis. Sympathetically-regulated lipid metab- stimulate the secretion of adrenocorticotropic hormoneolism and heat production in brown adipose tissue contrib- (ACTH), primarily in response to stress. CRF is also syn-utes to the non-shivering thermogenic component of stress-evoked hyperthermia and to energy expenditure aspects of thesized by neurons in other brain areas (Sakanaka et al.,body weight regulation. To identify potential central path- 1987) and central CRF receptor activation influences aways through which hypothalamic corticotropin releasing variety of functions including GI control (Tebbe et al.,factor influences brown adipose tissue thermogenesis, cor- 2005), anxiety and mood disorders (Bale and Vale, 2004),ticotropin releasing factor was microinjected into the lateral cardiovascular control (Fisher et al., 1983) and regulationventricle (i.c.v.) or into hypothalamic sites while recording of food intake and energy balance (Richard et al., 2000).sympathetic outflow to brown adipose tissue, brown adiposetissue temperature, expired CO2, heart rate and arterial pres- I.c.v. administration of CRF in rats produces a reduc-sure in urethane/chloralose-anesthetized, artificially-venti- tion in body weight (Arase et al., 1988; Buwalda et al.,lated rats. I.c.v. corticotropin releasing factor or corticotropin 1997), an increase in oxygen consumption (Strijbos et al.,releasing factor microinjection into the preoptic area or the 1992) and an activation of brown adipose tissue (BAT)dorsomedial hypothalamus, but not the paraventricular nu- thermogenesis (LeFeuvre et al., 1987). Studies to identifycleus of the hypothalamus, elicited sustained increases inbrown adipose tissue sympathetic nerve activity, brown adi- the central sites of action of CRF to stimulate metabolicpose tissue temperature, expired CO2 and heart rate. These energy consumption have indicated that neurons in thesympathetic responses to i.c.v. corticotropin releasing factor preoptic anterior hypothalamus (POA) are involved in thewere eliminated by inhibition of neuronal activity in the dor- BAT thermogenic stimulation by CRF (Egawa et al., 1990)somedial hypothalamus or in the raphe pallidus, a putative and that the PVN is a site at which CRF can reduce foodsite of sympathetic premotor neurons for brown adipose intake (Krahn et al., 1988).tissue, and were markedly reduced by microinjection of iono-tropic glutamate receptor antagonists into the dorsomedial Our understanding of the central pathways regulatinghypothalamus. The increases in brown adipose tissue sym- BAT sympathetic outflow and BAT thermogenesis andpathetic outflow, brown adipose tissue temperature and heart energy expenditure (Morrison, 2004b) has increased sincerate elicited from corticotropin releasing factor into the pre- these initial studies on central CRF-evoked responses.optic area were reversed by inhibition of neuronal discharge Specifically, anatomical and physiological studies have in-in dorsomedial hypothalamus. These data indicate that cor- dicated that the rostral raphe pallidus (RPa) area containsticotropin releasing factor release within the preoptic areaactivates a sympathoexcitatory pathway to brown adipose putative BAT sympathetic premotor neurons (Bamshad ettissue and to the heart, perhaps similar to that activated by al., 1999; Morrison et al., 1999; Cano et al., 2003; Naka-increased prostaglandin production in the preoptic area, that mura et al., 2005) and that neurons in the dorsomedialincludes neurons in the dorsomedial hypothalamus and in hypothalamus (DMH) are required for the increase in sym-*Corresponding author. Tel: ϩ1-503-418-2670; fax: ϩ1-503-418-2501. pathetic outflow to BAT evoked by microinjection of pros-E-mail address: (S. Morrison). taglandin E2 (PGE2) into the POA (Madden and Morrison,Abbreviations: ACTH, adrenocorticotropic hormone; AP, arterial pres- 2003; Zaretskaia et al., 2003; Nakamura et al., 2005), bysure; AP5, D-2-amino-5-phosphonovalerate; BAT, brown adipose tis-sue; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CRF, corticotropin the disinhibition of neurons in the lateral hypothalamusreleasing factor; DMH, dorsomedial hypothalamus; EAA, excitatory (LH) (Cerri and Morrison, 2005a) and by i.c.v. administra-amino acid; HR, heart rate; LH, lateral hypothalamic area; MAP, mean tion of the mu-opiate, fentanyl (Cao et al., 2004). In view ofarterial pressure; PGE2, prostaglandin E2; POA, preoptic area; PVN,paraventricular nucleus of the hypothalamus; RPa, raphe pallidus; the recently-identified significance of the synaptic integra-SNA, sympathetic nerve activity. tion sites in the RPa and the DMH in the control of BAT0306-4522/06$30.00ϩ0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO.doi:10.1016/j.neuroscience.2006.02.027 711
  11. 11. Neuroscience 135 (2005) 627– 638ACTIVATION OF LATERAL HYPOTHALAMIC NEURONS STIMULATESBROWN ADIPOSE TISSUE THERMOGENESISM. CERRIa,b AND S. F. MORRISONa* trating hormone (MCH)-containing neurons reside withina Neurological Sciences Institute, Oregon Health and Science Univer- the LH and both peptides are orexigenic. Orexin adminis-sity, 505 Northwest 185th Avenue, Beaverton, OR 97006, USA tration contributes to a behavioral state in which feedingb Dipartimento di Fisiologia Umana e Generale, Universita’ degli Studi occurs (Sakurai, 2002, 2005; Sakurai et al., 1998) and indi Bologna, Piazza di Porta S. Donato, 40126 Bologna, Italy which energy expenditure is stimulated (Wang et al., 2001). Chronic administration MCH leads to a form of obesity characterized by hyperphagia and a reduction inAbstract—The lateral hypothalamic area, containing orexinneurons, is involved in several aspects of autonomic regulation, thermogenesis in brown adipose tissue (BAT) (Ito et al.,including thermoregulation and energy expenditure. To deter- 2003), whereas MCH 1 receptor knock-out mice express amine if activation of lateral hypothalamic area neurons influ- hypermetabolic phenotype with a lower body fat massences sympathetically-regulated thermogenesis in brown adi- (Chen et al., 2002; Marsh et al., 2002).pose tissue, we microinjected bicuculline (120 pmol, 60 nl, Since lesions in the LH produce a profound anorexia,unilateral) into the lateral hypothalamic area in urethane/ loss of body weight and increase in metabolism ((Keeseychloralose-anesthetized, artificially-ventilated rats. Disinhibi- et al., 1984); see (Bernardis and Bellinger, 1996) for re-tion of neurons in lateral hypothalamic area evoked a signif- view), initial investigations on the role of LH neurons inicant increase (؉1309%) in brown adipose tissue sympa-thetic nerve activity accompanied by parallel increases in energy homeostasis suggested that they function to pro-brown adipose tissue temperature (؉2.0 °C), in expired CO2 mote energy storage by increasing the drive for food con-(؉0.6%), in heart rate (؉88 bpm) and in mean arterial pres- sumption and by inhibiting energy expenditure. The lattersure (؉11 mm Hg). Subsequent microinjections of glycine was suggested by the hyperthermia and the increase in the(30 nmol, 60 nl) to inhibit local neurons in raphe pallidus or in activity of the BAT that accompanied the weight lost fol-dorsomedial hypothalamus or of glutamate receptor antago- lowing LH lesions ((Corbett et al., 1988), although seenists into dorsomedial hypothalamus promptly reversed the (Park et al., 1988)). Since these responses could haveincreases in brown adipose tissue sympathetic nerve activ-ity, brown adipose tissue temperature and heart rate evoked arisen either from interruption of axons coursing throughby disinhibition of neurons in lateral hypothalamic area. We the lesion site or from eliminating the activity of neurons inconclude that neurons in the lateral hypothalamic area can LH, questions remain concerning the role of LH neurons ininfluence brown adipose tissue sympathetic nerve activity, the regulation of energy expenditure in BAT. The presentbrown adipose tissue thermogenesis and heart rate through study was initiated to determine the effects of cell-specificpathways that are dependent on the activation of neurons in activation of LH neurons on the sympathetically-regulateddorsomedial hypothalamus and raphe pallidus. © 2005 IBRO. thermogenesis of BAT.Published by Elsevier Ltd. All rights reserved.Key words: raphe pallidus, dorsomedial hypothalamus, bicu- EXPERIMENTAL PROCEDURESculline, orexin, sympathetic nerve activity, thermoregulation. General procedures Experiments were performed in accordance with the NationalNeurons in the lateral hypothalamus (LH) and perifornical Institutes of Health Guide for the Care and Use of Laboratoryregion have been implicated in the hypothalamic regulation Animals (NIH publication no. 80 –23, 1996) and under protocolsof energy homeostasis (Kalra et al., 1999), including both approved by the Institutional Animal Care and Use Committee offeeding and energy metabolism which, in turn, determine Oregon Health and Science University. The experimental design and anesthetic protocol minimized the number of animals usedthe level of stored energy and body weight. Populations of and prevented any indication of pain perception. Sprague–DawleyLH neurons also contribute to the control of the sleep– rats (nϭ23; 250 – 450 g) were obtained from Charles River, Inc.wake cycle (John et al., 2004; Piper et al., 2000) and to (Indianapolis, IN, USA) Animals were anesthetized i.v. with ure-cardiovascular responses (Shirasaka et al., 2002). Sepa- thane (0.8 g/kg) and chloralose (80 mg/kg) after induction with 4%rate populations of orexin-containing and melanin-concen- isoflurane in 100% O2. A femoral artery, a femoral vein and the trachea were cannulated for measurement of arterial pressure*Corresponding author. Tel: ϩ1-503-418-2670; fax: ϩ1-503-418-2501. (AP), drug injection and artificial ventilation, respectively. HeartE-mail address: (S. F. Morrison). rate (HR) was derived from the AP signal. After the animals wereAbbreviations: AP, arterial pressure; AP5, D-2-amino-5-phosphonovaler- positioned prone in a stereotaxic frame according to the approachate; BAT, brown adipose tissue; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DA, dorsal hypothalamic area; DMH, dorsomedial hypothala- of (Paxinos and Watson 1997) with the incisor bar at Ϫ4.0 mm andmus; EAA, excitatory amino acid; HR, heart rate; iDMH, dorsomedial with a spinal clamp on the T10 vertebra, they were paralyzed withhypothalamus ipsilateral; LH, lateral hypothalamic area; MCH, melanin- D-tubocurarine (0.3 mg initial dose, 0.1 mg/h supplements) andconcentrating hormone; PRV, pseudorabies virus; RPa, raphe pallidus; artificially ventilated with 100% O2 (50 cycles/min, tidal volume:SNA, sympathetic nerve activity. 3– 4.5 ml). Small adjustments in minute ventilation were made as0306-4522/05$30.00ϩ0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2005.06.039 627
  12. 12. Neuroscience Letters 383 (2005) 182–187 Changes in EEG activity and hypothalamic temperature as indices for non-REM sleep to REM sleep transitions Paolo Capitani a , Matteo Cerri b , Roberto Amici b , Francesca Baracchi b , Christine Ann Jones b , Marco Luppi b , Emanuele Perez b , Pier Luigi Parmeggiani b , Giovanni Zamboni b,∗ a Department of Electronics, Computer Science and Systems, Alma Mater Studiorum-University of Bologna, Italy b Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Piazza di Porta S. Donato, 2, I-40126 Bologna, Italy Received 18 February 2005; received in revised form 25 March 2005; accepted 3 April 2005Abstract A shift of physiological regulations from a homeostatic to a non-homeostatic modality characterizes the passage from non-NREM sleep(NREMS) to REM sleep (REMS). In the rat, an EEG index which allows the automatic scoring of transitions from NREMS to REMS has beenproposed: the NREMS to REMS transition indicator value, NIV [J.H. Benington et al., Sleep 17 (1994) 28–36]. However, such transitionsare not always followed by a REMS episode, but are often followed by an awakening. In the present study, the relationship between changesin EEG activity and hypothalamic temperature (Thy), taken as an index of autonomic activity, was studied within a window consisting of the60 s which precedes a state change from a consolidated NREMS episode. Furthermore, the probability that a transition would lead to REMSor wake was analysed. The results showed that, within this time window, both a modified NIV (NIV60 ) and the difference between Thy at thelimits of the window (ThyD ) were related to the probability of REMS onset. Both the relationship between the indices and the probability ofREMS onset was sigmoid, the latter of which saturated at a probability level around 50–60%. The efficacy for the prediction of successfultransitions from NREMS to REMS found using ThyD as an index supports the view that such a transition is a dynamic process where thephysiological risk to enter REMS is weighted at a central level.© 2005 Elsevier Ireland Ltd. All rights reserved.Keywords: Non-REM sleep to REM sleep transition; REM sleep; EEG activity; NIV60 ; Hypothalamic temperatureThe change in the behavioural state of non-REM sleep approximately 1 min have been observed in the cat, where(NREMS) to that of REM sleep (REMS) is a critical period, unit firing rate in the posterolateral cortex (lateral andsince physiological regulation shifts from a homeostatic suprasylvian gyrus) progressively increases from NREMSto a non-homeostatic modality [19,21]. We suggest that to REMS [15]. In the rat, cat and mouse, such a transition isthis transition from NREMS to REMS is controlled by usually characterized by specific changes in the electroen-integrative autonomic structures that encompass regulated cephalogram (EEG). These consist of successive short boutschanges occurring in anticipation of the event. This study (<10 s) of high-amplitude spindles from the anterior cerebralis concerned with the changes in two of the physiological cortex which are associated with a theta rhythm from thevariables that may be used to characterize such a transition. dorsal hippocampus [9,10,12]. This distinct pattern of the As far as cortical bioelectrical activity is concerned, it EEG has lead to the proposal that these brief periods identifyhas been shown that this transition does not occur abruptly, a sleep stage which is separate from both NREMS andbut is a smooth change which occurs between the two REMS and has been called either “the intermediate stage ofstates in which some of the features of NREMS gradually sleep” [11], “pre-REM sleep” [22] or “transition sleep” [14].lead into REMS. With respect to this, transitions lasting The frequency analysis of the EEG of the rat has shown that this transition (NREMS to REMS transition, NRT, [4]) ∗ Corresponding author. Tel.: +39 051 2091742; fax: +39 051 251731. lasts 30–60 s during which the EEG power density in the Delta E-mail address: (G. Zamboni). band progressively decreases, whilst that in both the Theta0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.neulet.2005.04.009
  13. 13. Cold Exposure and Sleep in the Rat: Effects on Sleep Architecture and theElectroencephalogramMatteo Cerri, MD, PhD1; Adrian Ocampo-Garces, MD, PhD2; Roberto Amici, MD1; Francesca Baracchi1; Paolo Capitani3; Christine Ann Jones, PhD1; Marco Luppi,PhD1; Emanuele Perez, MD1; Pier Luigi Parmeggiani, MD1; Giovanni Zamboni, MD1Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Italy; 2Programa de Fisiología y Biofísica, Instituto de1Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile; 3Department of Electronics, Computer Science and Systems, AlmaMater Studiorum-University of Bologna, Italy Study Objectives: Acute exposure to low ambient temperature modifies power during non-rapid eye movement sleep was decreased in animals the wake-sleep cycle due to stage-dependent changes in the capacity to exposed to the lowest ambient temperatures and increased during the first regulate body temperature. This study was carried out to make a sys- day of the recovery. In contrast, rapid eye movement sleep was greatly tematic analysis of sleep parameters during the exposure to different low depressed by cold exposure and showed an increase during the recovery. ambient temperatures and during the following recoveries at ambient tem- Both of these effects were dependent on the ambient temperature of the perature 24°C. exposure. Moreover, theta power was increased during rapid eye move- Design: Electroencephalographic activity, hypothalamic temperature, and ment sleep in both the exposure and the first day of the recovery. motor activity were studied during a 24-hour exposure to ambient temper- Conclusion: These findings show that sleep-stage duration and electro- atures ranging from 10°C to -10°C and for 4 days during the recovery. encephalogram power are simultaneously affected by cold exposure. The Setting: Laboratory of Physiological Regulation during the Wake-Sleep effects on rapid eye movement sleep appear mainly as changes in the Cycle, Department of Human and General Physiology, Alma Mater Stu- duration, whereas those on non-rapid eye movement sleep are shown by diorum-University of Bologna. changes in delta power. These effects are temperature dependent, and Subjects: Twenty-four male albino rats. the decrease of both parameters during the exposure is reciprocated by Interventions: Animals were implanted with electrodes for electroen- an increase in the subsequent recovery. cephalographic recording and a thermistor for measuring hypothalamic Key words: Low ambient temperature, sleep deprivation, NREM sleep, temperature. REM sleep, single REM sleep, sequential REM sleep, delta power den- Measurements and Results: Wake-sleep stage duration and the electro- sity, theta power density encephalographic spectral analysis performed by fast Fourier transform Citation: Cerri M; Ocampo-Garces A; Amici R et al. Cold exposure and were compared among baseline, exposure, and recovery conditions. The sleep in the rat: effects on sleep architecture and the electroencephalo- amount of non-rapid eye movement sleep was slightly depressed by cold gram. SLEEP 2005;28(6):694-705. exposure, but no rebound was observed during the recovery period. DeltaINTRODUCTION different low ambient temperatures was shown to primarily af- fect REM-sleep occurrence and that the observed decrease wasEXPOSURE TO AN AMBIENT TEMPERATURE OUTSIDE proportional to the ambient temperature of exposure, while more-THE APPROPRIATE THERMONEUTRAL RANGE CHANG- complex effects were observed on non-REM (NREM) sleep.1,2ES THE AMOUNT AND DISTRIBUTION OF THE DIFFER- With respect to the latter, “spindle sleep” was progressively de-ENT stages of the wake-sleep cycle in several different species.1-3 pressed at ambient temperatures below 0°C, while slow-waveThese changes may be a consequence of the differences in the sleep was maximally decreased at an ambient temperature of 5°Ccapacity to regulate body temperature across the different stages but increased toward control levels as the temperature was low-of the wake-sleep cycle.3,4 In particular, it has been shown that ered.1,2,7 This depression in sleep occurrence by the exposure tothermoregulatory responses like shivering, panting, and peripher- low ambient temperature has now been confirmed by many stud-al vasomotion are suppressed during rapid eye movement (REM) ies on different species.8-15sleep5 due to a change in the activity of the central thermostat at Exposure to low ambient temperature also clearly influencesthe preoptic-hypothalamic level.6 sleep occurrence when animals are allowed to recover at normal In early studies carried out in the cat, a short-term exposure to ambient temperature in the laboratory. In particular, a rebound of REM sleep, which was proportional to the degree of the previous REM sleep loss, has been observed in the cat.16 These results haveDisclosure Statement been confirmed in recent studies on the albino rat, in which it wasThis was not an industry supported study. Drs. Zamboni, Cerri, Ocampo- observed that both REM-sleep loss and the subsequent REM-sleepGarces, Amici, Baracchi, Capitani, Jones, Luppi, Perez, and Parmeggiani rebound were quantitatively related to the thermal load (durationhave indicated no financial conflicts of interest. of the exposure × decrease in ambient temperature with respectSubmitted for publication November 2004 to normal laboratory conditions).17-19 It has also been shown thatAccepted for publication February 2005 the amount of NREM sleep is less affected during recovery, sinceAddress correspondence to: Giovanni Zamboni, MD, Department of Human no substantial increase in its amount has been found in either theand General Physiology, Alma Mater Studiorum-University of Bologna,Piazza cat1,2 or the rat.14 However, an increase in the electroencephalo-P.ta S. Donato, 2, I-40126 Bologna, Italy; Tel: 39 051 2091742; Fax: 39 051 gram (EEG) power density in the delta band (0.75-4 Hz) during251731; E-mail: NREM sleep has been observed in the rat.14SLEEP, Vol. 28, No. 6, 2005 694 Cold Exposure and Sleep in the Rat—Cerri et al
  14. 14. Brain Research 1022 (2004) 62 – 70 Research report Specific changes in cerebral second messenger accumulation underline REM sleep inhibition induced by the exposure to low ambient temperature Giovanni Zamboni*, Christine Ann Jones, Rosa Domeniconi, Roberto Amici, Emanuele Perez, Marco Luppi, Matteo Cerri, Pier Luigi Parmeggiani Dipartimento di Fisiologia umana e generale, Universita di Bologna, Bologna, Italy ` Accepted 6 July 2004 Available online 11 August 2004Abstract In the rat the exposure to an ambient temperature (Ta) of À10 8C induces an almost total REM sleep deprivation that results in aproportional rebound in the following recovery at normal laboratory Ta when the exposure lasts for 24 h, but in a rebound much lower thanexpected when the exposure lasts 48 h. The possibility that this may be related to plastic changes in the nervous structures involved in thecontrol of thermoregulation and REM sleep has been investigated by measuring changes in the concentration of adenosine 3V:5V-cyclicmonophosphate (cAMP) and d-myo-inositol 1,4,5-trisphosphate (IP3) in the preoptic-anterior hypothalamic area (PO-AH), the ventromedialhypothalamic nucleus (VMH) and, as a control, the cerebral cortex (CC). Second messenger concentration was determined in animals eitherstimulated by being exposed to hypoxia, a depolarizing condition that induces maximal second messenger accumulation or unstimulated, atthe end of a 24-h and a 48-h exposure to À10 8C and also between 4 h 15 min and 4 h 30 min into recovery (early recovery). At the end ofboth exposure conditions, cAMP concentration significantly decreased in PO-AH-VMH, but did not change in CC, whilst changes in IP3concentration were similar in all these regions. The low cAMP concentration in PO-AH-VMH was concomitant with a significantly lowaccumulation in hypoxia. The normal capacity of cAMP accumulation was only restored in the early recovery following 24 h of exposure, butnot following 48 h of exposure, suggesting that this may be a biochemical equivalent of the REM sleep inhibition observed during 48 h ofexposure and which is carried over to the recovery.D 2004 Elsevier B.V. All rights reserved.Theme: Endocrine and autonomic regulationTopic: Osmotic and thermal regulationKeywords: Preoptic-anterior hypothalamic area; Adenosine cyclic monophosphate; Inositol trisphosphate; Low ambient temperature; REM sleep 1. Introduction The exposure to low ambient temperature (Ta) represents a physiological procedure for sleep deprivation which was Abbreviations: cAMP, adenosine 3V -cyclic monophosphate; CC, :5V first applied to the cat and was shown to affect REM sleepcerebral cortex; IP3, d-myo-inositol 1,4,5-trisphosphate; LC, locus coer- more selectively than NREM sleep [32]. In the rat, it hasuleus; PO-AH, preoptic-anterior hypothalamic area; Ta, ambient temper- been observed that the exposure to low Ta induces a REMature; VMH, ventromedial hypothalamic nucleus sleep loss and an immediate rebound which is proportional * Corresponding author. Dipartimento di Fisiologia umana e generalePiazza di Porta San Donato 2, 40127 Bologna BO, Italy. Tel.: +39 051 to the thermal load of exposure (Ta level by duration of2091742; fax: +39 051 251731. exposure) when animals were returned to recover at normal E-mail address: (G. Zamboni). laboratory Ta [2,3,4,15]. However, it has been observed that0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2004.07.002