Behavioral/Cognitive

The Inhibition of Neurons in the Central Nervous Pathwaysfor Thermoregulatory Cold Defense Induces a SuspendedAnimation State in the Rat

Matteo 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 Italy

The 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 � 0.74°C), heart rate (from 440 � 13 to207 � 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.

IntroductionSuspended animation is a temporary and fully reversible condi-tion characterized by hypometabolism and deep hypothermia,during which physiological functions are slowed down. In mam-mals, this condition spontaneously occurs under the form of tor-por and hibernation, which are triggered by environmentalfactors (Melvin and Andrews, 2009). The cellular and molecularmechanisms of natural suspended animation are still unknown(Carey et al., 2003), but since hypothermia is preceded by a met-abolic rate reduction (Heldmaier et al., 2004), it has been hypoth-

esized that two basic players take part in inducing a reduction inmetabolism: humoral factors (Andrews, 2007) and the CNS(Drew et al., 2007).

The intervention of humoral factors has recently been high-lighted by the induction of a suspended animation state in mice,a species where torpor occurs naturally, by the administration ofseveral substances interfering with cell metabolism (Scanlan etal., 2004; Blackstone et al., 2005; Gluck et al., 2006; Zhang et al.,2006). However, the translational outcomes of this approach(Lee, 2008) have been hampered, so far, by the failure to replicatethese results in nonhibernators (Haouzi et al., 2008, Zhang et al.,2009). Although the intervention of the CNS in determining nat-ural suspended animation remains largely unexplored (Drew etal., 2007), the increase in heat loss and the decrease in heat gen-eration that follow the chemical manipulation of the central ner-vous pathways for thermoregulatory cold defense (Morrison andNakamura, 2011) suggests that the reduction in metabolism mayalso be actively driven by the CNS.

A key area in the central nervous pathways for thermoregula-tory cold defense is the rostral ventromedial medulla (RVMM), aregion including the raphe pallidus (RPa) and the raphe magnus,where the putative sympathetic premotor neurons to the brownadipose tissue (BAT), the cutaneous blood vessel, and the heartare located (Cano et al., 2003). The activation of RPa neurons hasbeen shown to promote nonshivering (Morrison et al., 1999) andshivering (Nakamura and Morrison, 2011) thermogenesis, cuta-neous vasoconstriction (Blessing and Nalivaiko, 2001), and an

Received July 27, 2012; revised Dec. 19, 2012; accepted Dec. 20, 2012.Author contributions: M.C., G.Z., and R.A. designed research; M.C., M.M., D.T., and D.M. performed research; M.C.,

M.M., D.T., and M.L. analyzed data; M.C., E.P., G.Z., and R.A. wrote the paper.This work is supported by the Ministero dell’Universita e della Ricerca Scientifica (MIUR), Italy, (PRIN 2008, Project

2008FY7K9S).*D.T. and M.M. equally contributed to this work.The authors declare no competing financial interests.Correspondence should be addressed to Matteo Cerri, Department of Biomedical and NeuroMotor Sci-

ences, Alma Mater Studiorum–University of Bologna Piazza di Porta S. Donato 2, 40126 Bologna, Italy.E-mail: matteo.cerri@unibo.it.

M. Mastrotto’s present address: Department of Biological and Biomedical Sciences, Yale University, New Haven,CT 06520.

D. Tupone’s present address: Department of Neurological Surgery, Oregon Health and Science University, Port-land, OR 97239-3098.

D. Martelli’s present address: Systems Neurophysiology Division, Florey Institute of Neuroscience and MentalHealth, University of Melbourne, Parkville, Victoria 3010, Australia.

DOI:10.1523/JNEUROSCI.3596-12.2013Copyright © 2013 the authors 0270-6474/13/332984-10$15.00/0

2984 • The Journal of Neuroscience, February 13, 2013 • 33(7):2984 –2993

Waking and Sleeping following Water Deprivation in theRatDavide Martelli1,2, Marco Luppi1, Matteo Cerri1, Domenico Tupone1,3, Emanuele Perez1,

Giovanni Zamboni1*, Roberto Amici1

1 Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Bologna, Italy, 2 Systems Neurophysiology Division, Florey Neuroscience

Institutes, University of Melbourne, Melbourne, Australia, 3 Oregon National Primate Research Center, Oregon Health & Science University, Portland, Oregon, United States

of America

Abstract

Wake-sleep (W-S) states are affected by thermoregulation. In particular, REM sleep (REMS) is reduced in homeotherms undera thermal load, due to an impairment of hypothalamic regulation of body temperature. The aim of this work was to assesswhether osmoregulation, which is regulated at a hypothalamic level, but, unlike thermoregulation, is maintained across thedifferent W-S states, could influence W-S occurrence. Sprague-Dawley rats, kept at an ambient temperature of 24uC andunder 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 toassess: i) the maintenance of osmotic homeostasis (water and food consumption; changes in body weight and fluidcomposition); 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 dayof recovery, with the exception of urinary Ca++ that almost disappeared in WD, and increased to a high level in recovery. Asfar as the second set is concerned, WD was characterized by the maintenance of the daily oscillation of Thy and by adecrease 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 andrecovery, but EEG delta power, a sign of NREMS intensity, decreased in WD and increased in recovery. Our data suggest thatosmoregulation 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: � 2012 Martelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted 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.unibo.it/PortaleEn/Research/Services+for+teachers+and+researchers/National+Regional+and+Local+Funding/default.htm). The funders had no role in studydesign, 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.it

Introduction

Physiological regulation is known to be different during rapid

eye movement sleep (REMS) when compared to non rapid eye

movement sleep (NREMS) [1]. In particular, during NREMS a

stable autonomic outflow is observed in the presence of a fully

operant homeostatic control of physiological variables [1–4]. In

contrast, during REMS a high variability of the autonomic

outflow, which leads to large irregularities in arterial blood

pressure, heart rate, and respiratory rhythm, is concomitant with

an impairment of thermoregulation [1–4]. This impairment has

been considered to be a visible consequence of a change in

hypothalamic integrative activity, disabling, during REMS, the

autonomic feedbacks sustaining body homeostasis [1]. According-

ly, Wake-Sleep (W-S) states change when a tonic maintenance of

the hypothalamic regulation is needed, as it occurs during the

exposure to a low ambient temperature (Ta). In these conditions,

Wake increases, NREMS is variably affected and REMS is always

reduced or even suppressed in proportion to the Ta levels

[2,4,5,6].

However, the recent finding from our laboratory that, following

a central osmotic stimulation, the release of the antidiuretic

hormone arginine vasopressin (AVP) was kept at the same levels in

the different wake-sleep (W-S) states, indicates that hypothalamic

osmoregulation is not impaired during REMS [7]. This suggests

that the change in hypothalamic integrative activity in this sleep

stage should concern structures related to thermoregulation, rather

than the whole hypothalamus as previously hypothesized [1].

Since clarification regarding the issue of specificity of the

impairment in physiological regulation during REMS may be

relevant for the understanding of this sleep state, we sought to test

the observed independence of osmoregulation from autonomic

changes in sleep. To this end, REMS occurrence and overall W-S

regulation were assessed in rats during exposure to a three-day

water deprivation (WD) protocol. This condition is known to

constitute an osmotic challenge leading to the progressive

engagement of the whole set of mechanisms maintaining body

fluid homeostasis [8,9]. Moreover, since a REMS rebound was

observed during the recovery (R) period following cold exposure

[5,6,10,11,12,13], W-S assessment was continued for two days

after water was once again made freely available. Basically, two

sets of parameters were assessed: i) those concerning the

maintenance of osmotic homeostasis (water and food consump-

PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e46116

ECOTOXICOLOGYAND ENVIRONMENTAL TOXICOLOGY : NEW CONCEPTS, NEW TOOLS

Effects of chronic exposure to radiofrequencyelectromagnetic fields on energy balance in developing rats

Amandine Pelletier & Stéphane Delanaud &

Pauline Décima & Gyorgy Thuroczy & René de Seze &

Matteo Cerri & Véronique Bach & Jean-Pierre Libert &Nathalie Loos

Received: 4 July 2012 /Accepted: 16 October 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract The effects of radiofrequency electromagneticfields (RF-EMF) on the control of body energy balance indeveloping organisms have not been studied, despite theinvolvement of energy status in vital physiological functions.We examined the effects of chronic RF-EMF exposure(900 MHz, 1 Vm−1) on the main functions involved in bodyenergy homeostasis (feeding behaviour, sleep and thermoreg-ulatory processes). Thirteen juvenile male Wistar rats wereexposed to continuous RF-EMF for 5 weeks at 24 °C of airtemperature (Ta) and compared with 11 non-exposed animals.Hence, at the beginning of the 6th week of exposure, thefunctions were recorded at Ta of 24 °C and then at 31 °C.We showed that the frequency of rapid eye movement sleepepisodes 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 parameterswere dependent on Ta. At 31 °C, RF-EMF-exposed animalshad a significantly lower subcutaneous tail temperature(−1.21 °C) than controls at all sleep stages; this suggested

peripheral vasoconstriction, which was confirmed in an ex-periment with the vasodilatator prazosin. Exposure to RF-EMF also increased daytime food intake (+0.22 gh−1). Mostof the observed effects of RF-EMF exposure were dependenton Ta. Exposure to RF-EMF appears tomodify the functioningof vasomotor tone by acting peripherally through α-adrenoceptors. The elicited vasoconstriction may restrict bodycooling, whereas energy intake increases. Our results showthat RF-EMF exposure can induce energy-saving processeswithout strongly disturbing the overall sleep pattern.

Keywords Radiofrequency electromagnetic field . Sleep .

Feeding behaviour . Thermoregulation . Young rat

Introduction

Body energy balance is a relevant factor in growing organisms,since energy is required for vital functions, thermoregulationand tissue synthesis (in that order). The regulation of energybalance involves a complicated interaction between energyintake (feeding behaviour), energy saving (sleep), energy-dissipating mechanisms (vasomotricity) and energy produc-tion, all of which are controlled by hypothalamic structures(Blessing 2003; El Hajjaji et al. 2011; Himms-Hagen 1995).

Several studies have shown that these functions are incompetition; sleep and feeding cannot be performed simul-taneously, for example. In the rat, there is a positive corre-lation between meal size and the durations of both non-rapideye movement sleep (NREMS) and rapid eye movementsleep (REMS): the larger the energy intake, the longer therat will sleep (Danguir and Nicolaidis 1985). As far asthermal status and feeding behaviour are concerned, bodycooling and body heating stimulate and reduce food intake,respectively (De Vries et al. 1993; El Hajjaji et al. 2011).Thus, an animal in a cold environment consumes extra food

Responsible editor: Philippe Garrigues

A. Pelletier : S. Delanaud : P. Décima :V. Bach : J.-P. Libert :N. Loos (*)PériTox Laboratory (EA 4285-UMI01), Faculty of Medicine,Jules Verne University of Picardy (UPJV),3 rue des Louvels, CS 13602,80036 Amiens cedex 1, Francee-mail: nathalie.loos@u-picardie.fr

G. Thuroczy :R. de SezePériTOX Laboratory (EA 4285-UMI01), VIVA/TOXI,National Institute of Industrial Environment and Risks (INERIS),Parc ALATA BP2,60550 Verneuil-en-Halatte, France

M. CerriDepartment of Human and General Physiology,Alma Mater Studiorum-University of Bologna,Bologna, Italy

Environ Sci Pollut ResDOI 10.1007/s11356-012-1266-5

doi: 10.1111/j.1365-2869.2009.00810.x

Hypothalamic osmoregulation is maintained across the

wake–sleep cycle in the rat

MARCO LUPP I 1 , DAV IDE MARTELL I 1 , ROBERTO AMIC I 1 , FRANCESCA

BARACCH I 2 , MATTEO CERR I 1 , DAN IELA DENT ICO 1 , EMANUELE

PEREZ 1 and G IOVANN I ZAMBON I 11Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Bologna, Italy and 2Department of Human

Physiology, University of Milan, Milan, Italy

Accepted 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

INTRODUCTION

Rapid eye movement sleep (REMS) is characterized by

changes in physiological regulation, which differentiate this

sleep state from Wake and non-rapid eye movement sleep

(NREMS; Parmeggiani, 2005). In particular, thermoregulation

has been shown to be impaired during REMS in different

species (Heller, 2005; Parmeggiani, 2003), and this change has

been interpreted as the outcome of a more general impairment

in the hypothalamic integration of autonomic function, which

would explain the high degree of instability of the autonomic

activity during REMS (Parmeggiani, 1980, 1988, 1994).

The issue of generalizing the impairment of thermoregula-

tion during REMS to all hypothalamic integrative mechanisms

has not been addressed so far from an experimental point of

view. A way to address this problem is that of studying

regulations that have the highest degree of integration at the

hypothalamic level and that therefore display a more intense

disruption in case of a local impairment of integrative activity.

In line with this view, the aim of this work was to study the

regulation of body fluid osmolality during the different wake–

sleep (W–S) states, as it represents a function that is almost

completely integrated at the hypothalamic level in mammals

(Denton et al., 1996).

In these species, the most efficient defense of osmolality is

provided by the release of arginine-vasopressin (AVP), the

anti-diuretic hormone, by hypothalamic magnocellular neu-

rons of the supraoptic nucleus (SON) and paraventricular

Correspondence: Giovanni Zamboni, MD, Department of Human and

General Physiology, Alma Mater Studiorum-University of Bologna,

Piazza P.ta S. Donato, 2, I-40126 Bologna, Italy. Tel.: +39 051

2091742; fax: +39 051 2091737; e-mail: giovanni.zamboni@unibo.it

J. Sleep Res. (2010) 19, 394–399 Osmoregulation and sleep

394 � 2010 European Sleep Research Society

CUTANEOUS VASODILATION ELICITED BY DISINHIBITION OF THECAUDAL PORTION OF THE ROSTRAL VENTROMEDIAL MEDULLA OFTHE FREE-BEHAVING RAT

M. CERRI,* G. ZAMBONI, D. TUPONE, D. DENTICO,M. LUPPI, D. MARTELLI, E. PEREZ AND R. AMICI

Dipartimento di Fisiologia Umana e Generale, Alma Mater StudiorumUniversità di Bologna, Piazza di Porta S. Donato 2, 40126, Italy

Abstract—Putative sympathetic premotor neurons control-ling cutaneous vasomotion are contained within the rostralventromedial medulla (RVMM) between levels corresponding,rostrally, to the rostral portion of the nucleus of the facialnerve (RVMM(fn)) and, caudally, to the rostral pole of theinferior olive (RVMM(io)). Cutaneous vasoconstrictor premo-tor neurons in the RVMM(fn) play a major role in mediatingthermoregulatory changes in cutaneous vasomotion that reg-ulate heat loss. To determine the role of neurons in theRVMM(io) in regulating cutaneous blood flow, we examinedthe changes in the tail and paw skin temperature of free-behaving rats following chemically-evoked changes in theactivity of neurons in the RVMM(io). Microinjection of theGABAA agonist, muscimol, within either the RVMM(fn) orthe RVMM(io) induced a massive peripheral vasodilation; mi-croinjection of the GABAA antagonist bicuculline methiodidewithin the RVMM(fn) reversed the increase in cutaneousblood flow induced by warm exposure and, unexpectedly,disinhibition of RVMM(io) neurons produced a rapid cutane-ous vasodilation. We conclude that the tonically-active neu-rons driving cutaneous vasoconstriction, likely sympatheticpremotor neurons previously described in the RVMM(fn), arealso located in the RVMM(io). However, in the RVMM(io),these are accompanied by a population of neurons that re-ceives a tonically-active GABAergic inhibition in the con-scious animal and that promotes a cutaneous vasodilationupon relief of this inhibition. Whether the vasodilator neuronslocated in the RVMM(io) play a role in thermoregulation re-mains to be determined. © 2010 IBRO. Published by ElsevierLtd. All rights reserved.

Key words: infrared thermography, thermoregulation, sym-pathetic nervous system, cutaneous vasomotion, muscimol,bicuculline methiodide.

The rostral ventromedial medulla (RVMM) contains popu-lations of sympathetic premotor neurons controlling sev-eral autonomic functions, including cutaneous vasomotion(Smith et al., 1998; Nagashima et al., 2000; Blessing andNalivaiko, 2001; Nakamura et al., 2004), brown adipose

tissue (BAT) thermogenesis (Morrison, 2001; Cano et al.,2003; Nakamura et al., 2005) and cardiac rate (Cao andMorrison, 2003). Antagonism of GABAA receptors in theRVMM produces an increase in body temperature in theanaesthetized animal through both a peripheral vasocon-striction (Blessing and Nalivaiko, 2001) and an increase inBAT thermogenesis (Morrison et al., 1999). Conversely,the injection into the same area of a GABAA agonist leadsto vasodilation of the tail skin in the anaesthetized rat(Blessing and Nalivaiko, 2001), and to hypothermia (Za-retsky et al., 2003) and tail vasodilation (Vianna et al.,2008) in conscious rats.

Studies employing injections of the transneuronal ret-rograde tracer, pseudorabies virus within the rat tail arterywall, have localized putative sympathetic premotor neu-rons controlling cutaneous blood vessels throughout theRVMM, between the levels corresponding to the rostralportion of nucleus of the facial nerve and the rostral portionof the inferior olive (Smith et al., 1998; Nakamura et al.,2004; Toth et al., 2006). Interestingly, a relative differ-ence in neuronal phenotype has been suggested betweenthe rostral portion of the RVMM, containing more putativeglutamatergic neurons expressing the vesicular glutamatetransporter, VGLUT3, and the caudal portion of the RVMM,where more serotoninergic neurons have been identified(Nakamura et al., 2004; Stornetta et al., 2005; Toth et al.,2006). From both anatomical and physiological evidence, ithas been proposed that the neural substrate in the RVMMfor the control of cutaneous vasomotion is represented bya set of VGLUT3-positive, glutamatergic neurons directlyprojecting to the intermediolateral column (IML) of thespinal cord (Nakamura et al., 2004). These data have ledto a theoretical paradigm in which sympathetic outflow tocutaneous blood vessels is proportional to the level ofactivity of the RVMM glutamatergic sympathetic premotorneurons (Nakamura et al., 2004).

The evaluation of the physiological role of RVMM neu-rons controlling cutaneous blood flow has been until nowlimited to the rostral portion of the RVMM, within the rostro-caudal level of the nucleus of the facial nerve (RVMM(fn))(Tanaka et al., 2002; Ootsuka and Blessing, 2006), wherethe more VGLUT3 positive neurons are located. No dataare available on the role of the more caudal portion ofRVMM neurons, within the rostro-caudal section of therostral pole of the inferior olive (RVMM(io)), in controllingcutaneous vasomotion.

The aim of the present study is a more extensivecharacterization of the RVMM physiological role in control-ling cutaneous vasomotion, with special focus on the cau-

*Corresponding author. Tel: �39-051-2091756; fax: �39-051-2091737.E-mail address: matteo.cerri@unibo.it (M. Cerri).Abbreviations: AP, arterial pressure; BAT, brown adipose tissue; EKG,electrocardiogram; HR, heart rate; IML, intermediolateral nucleus ofthe spinal cord; RVMM, rostral ventromedial medulla; RVMM(fn), ros-tral ventromedial medulla at the level of the facial nucleus; RVMM(io),rostral ventromedial medulla at the level of the rostral inferior olivarynucleus; Ta, ambient temperature; Thy, hypothalamic temperature;Tpaw, paw temperature; Ttail, tail temperature.

Neuroscience 165 (2010) 984–995

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2009.10.068

984

BEHAVIORAL NEUROSCIENCE

c-Fos expression in preoptic nuclei as a marker of sleeprebound in the rat

Daniela Dentico,1 Roberto Amici,1 Francesca Baracchi,1,2 Matteo Cerri,1 Elide Del Sindaco,1 Marco Luppi,1

Davide Martelli,1 Emanuele Perez1 and Giovanni Zamboni11Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Piazza P.ta S. Donato, 2, I-40126Bologna, Italy2Research Division, Department of Anesthesiology, University of Michigan, Ann Arbor, MI, USA

Keywords: c-Fos, cold exposure, median preoptic nucleus, P-CREB, ventrolateral preoptic nucleus

Abstract

Thermoregulation 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.

Introduction

The preoptic area (POA) is known to be a key structure in theregulation of body temperature (Romanovsky, 2007), and has beenclearly recognized as a sleep-promoting site (Szymusiak et al., 2007),probably representing the diencephalic substrate of the integrationbetween thermoregulation and sleep-related processes (Parmeggiani,2003). A relevant feature of the POA is that almost 25% of neuronstherein increase their activity at sleep onset and ⁄ or during sleepoccurrence (Szymusiak et al., 2007). It has been proposed that twoPOA subregions, the ventrolateral preoptic nucleus (VLPO) and themedian preoptic nucleus (MnPO), may have a key role in sleep-relatedprocesses on the basis of the results of unit recording (Szymusiaket al., 1998; Suntsova et al., 2002, 2007), anatomical tracer (Sherinet al., 1996, 1998; Steininger et al., 2001; Uschakov et al., 2007) andimmunohistochemical studies (analysis of c-Fos expression) (Morgan& Curran, 1991; Sherin et al., 1996; Gong et al., 2000, 2004). Sleepdeprivation studies have suggested that, in both nuclei, c-Fosactivation is related to sleep occurrence and ⁄ or to an increase insleep pressure (Gong et al., 2004; Gvilia et al., 2006a,b).

Exposure to low ambient temperature (Ta) represents a useful toolfor inducing physiological sleep deprivation, which is followed by asleep rebound after a return to normal laboratory conditions(Parmeggiani, 2003). The thermoregulatory impairment that charac-terizes rapid eye movement sleep (REMS) makes cold exposureparticularly challenging for REMS (Parmeggiani, 2003; Heller, 2005).In particular, during prolonged exposure to very low Tas, REMSpressure was shown to increase dramatically, leading to an intenseREMS rebound during the following recovery (Amici et al., 1994,1998, 2008; Cerri et al., 2005). Extreme cold is apparently lesschallenging for non-rapid eye movement sleep (NREMS), as, under a24-h exposure to a Ta of )10 �C protocol, the changes in both NREMSamount and the power density in the delta band of the electroenceph-alogram were smaller than those observed in REMS amount, duringboth the exposure and the following recovery (Cerri et al., 2005).In the present study, in order to better assess the role of the VLPO

and MnPO in sleep-related processes under a long-term physiologicalsleep deprivation protocol, c-Fos expression was studied duringprolonged exposure to a Ta of )10 �C and the subsequent earlyrecovery at laboratory Ta. In addition, as prolonged exposure to a Ta of)10 �C was shown to dampen the maximum accumulation capacity ofthe second messenger cAMP at the POA level (Zamboni et al., 1996,1999, 2004), the expression of the phosphorylated form of the

Correspondence: Dr Giovanni Zamboni, as above.E-mail: giovanni.zamboni@unibo.it

Received 4 December 2008, revised 11 June 2009, accepted 16 June 2009

European Journal of Neuroscience, Vol. 30, pp. 651–661, 2009 doi:10.1111/j.1460-9568.2009.06848.x

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

European Journal of Neuroscience

doi: 10.1111/j.1365-2869.2008.00658.x

Cold exposure impairs dark-pulse capacity to induce REM sleep

in the albino rat

FRANCESCA BARACCH I 1 , 2 , G IOVANN I ZAMBON I 1 , MATTEO CERR I 1 ,

E L IDE DEL S INDACO 1 , DAN IELA DENT ICO 1 , CHR I ST INE ANN JONES 1 ,

MARCO LUPP I 1 , EMANUELE PEREZ 1 and ROBERTO AMIC I 11Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Italy and 2Department of Anesthesiology,

Research Division, University of Michigan, Ann Arbor, USA

Accepted 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.

k e y w o r d s dark pulse, low ambient temperature, non-REM sleep to REM sleep

transition, preotpic-anterior hypothalamus, REM sleep, REM sleep homeostasis

INTRODUCTION

The wake–sleep cycle consists of the alternation of three

different states, Wake, non-REM sleep (NREMS) and REM

sleep (REMS), which are usually identified on the basis of the

level of brain cortical and muscle activity. From a physiolog-

ical point of view, the major feature that differentiates REMS

from both Wake and NREMS consists of an operational

change in physiological regulation that shifts from a homeo-

static to a non-homeostatic modality during REMS (Par-

meggiani, 2005). Such a shift is more relevant for a regulation,

such as thermoregulation, that needs a high degree of

integration and largely depends on the activity of regulatory

structures of the hypothalamus (Parmeggiani, 2003). Thus,

REMS occurrence needs to be finely regulated, since it

represents a physiological challenge for the organism, partic-

ularly when under unfavourable ambient conditions, such as

during the exposure to a low ambient temperature (Ta).

In different species, the passage from NREMS to REMS has

been shown not to occur abruptly (Gottesmann, 1996;

Correspondence: Roberto Amici, Department of Human and General

Physiology, Alma Mater Studiorum-University of Bologna, Piazza

P.ta S. Donato, 2 I-40126 Bologna, Italy. Tel.: +39-051-2091735; fax:

+39-051-2091737; e-mail: roberto.amici@unibo.it

J. Sleep Res. (2008) 17, 166–179 Sleep in animals

166 � 2008 European Sleep Research Society

SLEEP, Vol. 31, No. 5, 2008 708

MANY STUDIES HAVE SHOWN THAT A SLEEP DEFICIT INDUCES A SUBSEQUENT INCREASE IN THE DURA-TION AND/OR IN THE INTENSITY OF SLEEP AND THAT the occurrence of sleep reduces sleep propensity. The outcome of the regulation of such a balance between sleep and wake has been addressed as “sleep homeostasis”.1,2

The major hindrance in a quantitative approach to sleep homeostasis lies in the fact that sleep consists of two different states, NREM sleep (NREMS) and REM sleep (REMS), which cyclically alternate on an ultradian basis. In particular: (1) it is not possible to carry out a selective NREMS deprivation with-out interfering with REMS occurrence; (2) selective REMS deprivation procedures have been shown to affect to some ex-tent the quality of NREMS during deprivation3,4; (3) a complex interaction between NREMS and REMS rebounds has been observed following different sleep deprivation protocols5,6; and (4) NREMS and REMS regulation are influenced differently by circadian rhythmicity.7

In spite of this, many different “short-term” (minutes/hours) or “long-term” (hours/days) sleep deprivation studies seeking a homeostatically regulated sleep parameter have shown that NREMS is substantially regulated in terms of intensity, and REMS in terms of duration.2 However, some contradictory as-pects arising from “extended” (days/weeks) sleep deprivation studies and/or from the comparison of animal and human stud-ies still leave this topic open to discussion.2,8-11

As far as NREMS is concerned, the power density in the delta band (approximately, 0.5-4.5 Hz) of the electroencepha-logram (EEG), not NREMS duration, is considered to be the homeostatically regulated parameter in NREMS and is com-monly taken as an index of NREMS intensity.1,2,12 Such a regu-lation appears to be disrupted following an extended period of either sleep deprivation or sleep restriction.9,13

REMS appears to be precisely regulated in terms of its dura-tion on both a short-term and long-term basis. The short-term 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-rectly related to the duration of the preceding REMS episode, but not to the duration of the subsequent REMS episode.14-17 The long-term component of REMS regulation is expressed in the total amount of REMS during the days following a total sleep or selective REM sleep deprivation. A precise REMS con-servation has been observed in both the cat and the rat, since the rebound in REMS has been found to be proportional to the total loss of REMS during the deprivation period. 11,18-22 This precise quantitative regulation of total REMS amount may not occur following an extended period of sleep deprivation or sleep restriction.9,13,23 Whereas changes in EEG power are an

REM SlEEp

Cold 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, MD1

1Department 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- Research Division, University of Michigan, Ann Arbor, MI

Study Objectives: Exposure to low ambient temperature (Ta) depress-es REM sleep (REMS) occurrence. In this study, both short and long-term homeostatic aspects of REMS regulation were analyzed during cold exposure and during subsequent recovery at Ta 24°C.Design: EEG activity, hypothalamic temperature, and motor activ-ity were studied during a 24-h exposure to Tas ranging from 10°C to –10°C and for 4 days during recovery.Setting: Laboratory of Physiological Regulation during the Wake-Sleep Cycle, Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna.Subjects: 24 male albino rats.Interventions: Animals were implanted with electrodes for EEG re-cording and a thermistor to measure hypothalamic temperature.Measurements and Results: REMS occurrence decreased propor-tionally with cold exposure, but a fast compensatory REMS rebound occurred during the first day of recovery when the previous loss went

beyond a “fast rebound” threshold corresponding to 22% of the daily REMS need. A slow REMS rebound apparently allowed the animals to fully restore the previous REMS loss during the following 3 days of recovery.Conclusion: Comparing the present data on rats with data from ear-lier studies on cats and humans, it appears that small mammals have less tolerance for REMS loss than large ones. In small mammals, this low tolerance may be responsible on a short-term basis for the shorter wake-sleep cycle, and on long-term basis, for the higher percentage of REMS that is quickly recovered following REMS deprivation.Keywords: REM sleep, low ambient temperature, REM sleep homeo-stasis, REM sleep rebound, body size, theta power density.Citation: Amici R; Cerri M; Ocampo-Garcés A; Baracchi F; Dentico D; Jones CA; Luppi M; Perez E; Parmeggiani PL; Zamboni G. Cold ex-posure and sleep in the rat: REM sleep homeostasis and body size. SLEEP 2008;31(5):708-715.

Disclosure StatementThis was not an industry supported study. Dr. Cerri has received research support from the European Sleep Research Society through a sponsor-ship from Sanofi-Aventis. The other authors have indicated no financial conflicts of interest.

Submitted for publication July, 2007Accepted for publication December, 2007Address correspondence to: Roberto Amici, MD, Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Piazza P.ta S. Donato, 2, I-40126 Bologna, Italy; Tel: +39-051-2091735; Fax: +39-051-2091737; E-mail: roberto.amici@unibo.it

REM Sleep Homeostasis and Body Size—Amici et al

Available online at www.sciencedirect.com

Behavioural Brain Research 187 (2008) 254–261

Research report

Lithium affects REM sleep occurrence, autonomic activityand brain second messengers in the rat�

Christine Ann Jones ∗, Emanuele Perez, Roberto Amici, Marco Luppi,Francesca Baracchi, Matteo Cerri, Daniela Dentico, Giovanni Zamboni

Dipartimento di Fisiologia umana e generale, Universita di Bologna, Bologna, Italy

Received 5 July 2006; received in revised form 7 August 2007; accepted 17 September 2007Available online 20 September 2007

Abstract

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 cortex

1. Introduction

Lithium is traditionally used in bipolar disorder to preventthe recurrence of episodes of mania [6] and also appears tobe very effective in the management of acute manic episodes[26,27,35]. A common symptom of these mood disorders isinsomnia [8], but a consistent alteration of sleep appears to affectthe first REM episode, which occurs at a latency shorter thanthat observed in normal individuals [7]. Although a shorten-ing of REM sleep latency is seen in other psychopathologicaldisturbances [38], it has been observed that the normalizationof REM sleep occurrence is concomitant with a symptomaticimprovement in depressed patients [8]. Likewise, the adminis-tration of lithium to manic depressive patients increases REMsleep latency [11].

� Supported by a grant from MIUR, Italy.∗ Corresponding author at: Dipartimento di Fisiologia umana e generale,

Piazza di Porta San Donato 2, 40126 Bologna, BO, Italy. Tel.: +39 051 2091726;fax: +39 051 2091737.

E-mail address: christineann.jones@unibo.it (C.A. Jones).

This study has been originated from two sets of separateexperimental results following REM sleep deprivation inducedby exposure to a very low ambient temperature (Ta) for 48 h.The first set of results showed that early recovery was character-ized by a rebound of REM sleep occurring with a longer delayand a lower rate than that observed for deprivations induced byshorter exposures, that is, characterized by a smaller REM sleeploss [3]. The second set showed that these changes in REM sleeprecovery were concomitant with changes in the brain accumula-tion capacity of second messenger: (i) the capacity to accumulateadenosine 3′:5′ cyclic monophosphate (cAMP) was significantlyreduced in the preoptic-anterior hypothalamic area (PO-AH), thethermoregulatory region involved in the control of the negativeinteraction between sleep and homeothermia, but not in the cere-bral cortex (CC), whose bioelectrical activity is used to classifysleep stages; (ii) the capacity to accumulate 1,4,5-trisphosphate(IP3) significantly increased in both PO-AH and CC [48].

Since several observations show that cAMP and IP3 brainlevels are influenced by lithium [15,22,34], we sought to studywhether the similarity of changes in REM sleep occurrencecaused by exposure to low Ta and by the administration of the

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbr.2007.09.017

CORTICOTROPIN RELEASING FACTOR INCREASES IN BROWNADIPOSE TISSUE THERMOGENESIS AND HEART RATE THROUGHDORSOMEDIAL HYPOTHALAMUS AND MEDULLARYRAPHE PALLIDUS

M. CERRIa,b AND S. F. MORRISONa*aNeurological Sciences Institute, Oregon Health & Science University,505 Northwest 185th Avenue, Beaverton, OR 97006, USAbDipartimento di Fisiologia Umana e Generale Universita’ degli Studi diBologna Piazza di Porta S. Donato, 40126, Bologna, Italy

Abstract—Corticotropin releasing factor, acting at hypotha-lamic corticotropin releasing factor receptors, contributes tothe neural signaling pathways mediating stress-related re-sponses, as well as those involved in maintaining energybalance homeostasis. Sympathetically-regulated lipid metab-olism and heat production in brown adipose tissue contrib-utes to the non-shivering thermogenic component of stress-evoked hyperthermia and to energy expenditure aspects ofbody weight regulation. To identify potential central path-ways through which hypothalamic corticotropin releasingfactor influences brown adipose tissue thermogenesis, cor-ticotropin releasing factor was microinjected into the lateralventricle (i.c.v.) or into hypothalamic sites while recordingsympathetic outflow to brown adipose tissue, brown adiposetissue temperature, expired CO2, heart rate and arterial pres-sure in urethane/chloralose-anesthetized, artificially-venti-lated rats. I.c.v. corticotropin releasing factor or corticotropinreleasing factor microinjection into the preoptic area or thedorsomedial hypothalamus, but not the paraventricular nu-cleus of the hypothalamus, elicited sustained increases inbrown adipose tissue sympathetic nerve activity, brown adi-pose tissue temperature, expired CO2 and heart rate. Thesesympathetic responses to i.c.v. corticotropin releasing factorwere eliminated by inhibition of neuronal activity in the dor-somedial hypothalamus or in the raphe pallidus, a putativesite of sympathetic premotor neurons for brown adiposetissue, and were markedly reduced by microinjection of iono-tropic glutamate receptor antagonists into the dorsomedialhypothalamus. The increases in brown adipose tissue sym-pathetic outflow, brown adipose tissue temperature and heartrate elicited from corticotropin releasing factor into the pre-optic area were reversed by inhibition of neuronal dischargein dorsomedial hypothalamus. These data indicate that cor-ticotropin releasing factor release within the preoptic areaactivates a sympathoexcitatory pathway to brown adiposetissue and to the heart, perhaps similar to that activated byincreased prostaglandin production in the preoptic area, thatincludes neurons in the dorsomedial hypothalamus and in

the raphe pallidus. © 2006 Published by Elsevier Ltd onbehalf of IBRO.

Key words: sympathetic nerve activity, energy balance,stress, hyperthermia.

Corticotropin releasing factor (CRF), identified in 1981(Vale et al., 1981), is a neuropeptide hormone synthesizedby neurons in the parvocellular paraventricular nucleus(PVN) of the hypothalamus that acts on pituitary cells tostimulate the secretion of adrenocorticotropic hormone(ACTH), primarily in response to stress. CRF is also syn-thesized by neurons in other brain areas (Sakanaka et al.,1987) and central CRF receptor activation influences avariety of functions including GI control (Tebbe et al.,2005), anxiety and mood disorders (Bale and Vale, 2004),cardiovascular control (Fisher et al., 1983) and regulationof food intake and energy balance (Richard et al., 2000).

I.c.v. administration of CRF in rats produces a reduc-tion in body weight (Arase et al., 1988; Buwalda et al.,1997), an increase in oxygen consumption (Strijbos et al.,1992) and an activation of brown adipose tissue (BAT)thermogenesis (LeFeuvre et al., 1987). Studies to identifythe central sites of action of CRF to stimulate metabolicenergy consumption have indicated that neurons in thepreoptic anterior hypothalamus (POA) are involved in theBAT thermogenic stimulation by CRF (Egawa et al., 1990)and that the PVN is a site at which CRF can reduce foodintake (Krahn et al., 1988).

Our understanding of the central pathways regulatingBAT sympathetic outflow and BAT thermogenesis andenergy expenditure (Morrison, 2004b) has increased sincethese initial studies on central CRF-evoked responses.Specifically, anatomical and physiological studies have in-dicated that the rostral raphe pallidus (RPa) area containsputative BAT sympathetic premotor neurons (Bamshad etal., 1999; Morrison et al., 1999; Cano et al., 2003; Naka-mura et al., 2005) and that neurons in the dorsomedialhypothalamus (DMH) are required for the increase in sym-pathetic outflow to BAT evoked by microinjection of pros-taglandin E2 (PGE2) into the POA (Madden and Morrison,2003; Zaretskaia et al., 2003; Nakamura et al., 2005), bythe disinhibition of neurons in the lateral hypothalamus(LH) (Cerri and Morrison, 2005a) and by i.c.v. administra-tion of the mu-opiate, fentanyl (Cao et al., 2004). In view ofthe recently-identified significance of the synaptic integra-tion sites in the RPa and the DMH in the control of BAT

*Corresponding author. Tel: �1-503-418-2670; fax: �1-503-418-2501.E-mail address: morrisos@ohsu.edu (S. Morrison).Abbreviations: ACTH, adrenocorticotropic hormone; AP, arterial pres-sure; AP5, D-2-amino-5-phosphonovalerate; BAT, brown adipose tis-sue; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CRF, corticotropinreleasing factor; DMH, dorsomedial hypothalamus; EAA, excitatoryamino acid; HR, heart rate; LH, lateral hypothalamic area; MAP, meanarterial pressure; PGE2, prostaglandin E2; POA, preoptic area; PVN,paraventricular nucleus of the hypothalamus; RPa, raphe pallidus;SNA, sympathetic nerve activity.

Neuroscience 140 (2006) 711–721

0306-4522/06$30.00�0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO.doi:10.1016/j.neuroscience.2006.02.027

711

ACTIVATION OF LATERAL HYPOTHALAMIC NEURONS STIMULATESBROWN ADIPOSE TISSUE THERMOGENESIS

M. CERRIa,b AND S. F. MORRISONa*aNeurological Sciences Institute, Oregon Health and Science Univer-sity, 505 Northwest 185th Avenue, Beaverton, OR 97006, USAbDipartimento di Fisiologia Umana e Generale, Universita’ degli Studidi Bologna, Piazza di Porta S. Donato, 40126 Bologna, Italy

Abstract—The lateral hypothalamic area, containing orexinneurons, is involved in several aspects of autonomic regulation,including thermoregulation and energy expenditure. To deter-mine if activation of lateral hypothalamic area neurons influ-ences sympathetically-regulated thermogenesis in brown adi-pose tissue, we microinjected bicuculline (120 pmol, 60 nl,unilateral) into the lateral hypothalamic area in urethane/chloralose-anesthetized, artificially-ventilated rats. Disinhibi-tion of neurons in lateral hypothalamic area evoked a signif-icant increase (�1309%) in brown adipose tissue sympa-thetic nerve activity accompanied by parallel increases inbrown adipose tissue temperature (�2.0 °C), in expired CO2

(�0.6%), in heart rate (�88 bpm) and in mean arterial pres-sure (�11 mm Hg). Subsequent microinjections of glycine(30 nmol, 60 nl) to inhibit local neurons in raphe pallidus or indorsomedial hypothalamus or of glutamate receptor antago-nists into dorsomedial hypothalamus promptly reversed theincreases in brown adipose tissue sympathetic nerve activ-ity, brown adipose tissue temperature and heart rate evokedby disinhibition of neurons in lateral hypothalamic area. Weconclude that neurons in the lateral hypothalamic area caninfluence brown adipose tissue sympathetic nerve activity,brown adipose tissue thermogenesis and heart rate throughpathways that are dependent on the activation of neurons indorsomedial hypothalamus and raphe pallidus. © 2005 IBRO.Published by Elsevier Ltd. All rights reserved.

Key words: raphe pallidus, dorsomedial hypothalamus, bicu-culline, orexin, sympathetic nerve activity, thermoregulation.

Neurons in the lateral hypothalamus (LH) and perifornicalregion have been implicated in the hypothalamic regulationof energy homeostasis (Kalra et al., 1999), including bothfeeding and energy metabolism which, in turn, determinethe level of stored energy and body weight. Populations ofLH neurons also contribute to the control of the sleep–wake cycle (John et al., 2004; Piper et al., 2000) and tocardiovascular responses (Shirasaka et al., 2002). Sepa-rate populations of orexin-containing and melanin-concen-

trating hormone (MCH)-containing neurons reside withinthe LH and both peptides are orexigenic. Orexin adminis-tration contributes to a behavioral state in which feedingoccurs (Sakurai, 2002, 2005; Sakurai et al., 1998) and inwhich energy expenditure is stimulated (Wang et al.,2001). Chronic administration MCH leads to a form ofobesity characterized by hyperphagia and a reduction inthermogenesis in brown adipose tissue (BAT) (Ito et al.,2003), whereas MCH 1 receptor knock-out mice express ahypermetabolic phenotype with a lower body fat mass(Chen et al., 2002; Marsh et al., 2002).

Since lesions in the LH produce a profound anorexia,loss of body weight and increase in metabolism ((Keeseyet al., 1984); see (Bernardis and Bellinger, 1996) for re-view), initial investigations on the role of LH neurons inenergy homeostasis suggested that they function to pro-mote energy storage by increasing the drive for food con-sumption and by inhibiting energy expenditure. The latterwas suggested by the hyperthermia and the increase in theactivity of the BAT that accompanied the weight lost fol-lowing LH lesions ((Corbett et al., 1988), although see(Park et al., 1988)). Since these responses could havearisen either from interruption of axons coursing throughthe lesion site or from eliminating the activity of neurons inLH, questions remain concerning the role of LH neurons inthe regulation of energy expenditure in BAT. The presentstudy was initiated to determine the effects of cell-specificactivation of LH neurons on the sympathetically-regulatedthermogenesis of BAT.

EXPERIMENTAL PROCEDURES

General procedures

Experiments were performed in accordance with the NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals (NIH publication no. 80–23, 1996) and under protocolsapproved by the Institutional Animal Care and Use Committee ofOregon Health and Science University. The experimental designand anesthetic protocol minimized the number of animals usedand prevented any indication of pain perception. Sprague–Dawleyrats (n�23; 250–450 g) were obtained from Charles River, Inc.(Indianapolis, IN, USA) Animals were anesthetized i.v. with ure-thane (0.8 g/kg) and chloralose (80 mg/kg) after induction with 4%isoflurane in 100% O2. A femoral artery, a femoral vein and thetrachea were cannulated for measurement of arterial pressure(AP), drug injection and artificial ventilation, respectively. Heartrate (HR) was derived from the AP signal. After the animals werepositioned prone in a stereotaxic frame according to the approachof (Paxinos and Watson 1997) with the incisor bar at �4.0 mm andwith a spinal clamp on the T10 vertebra, they were paralyzed withD-tubocurarine (0.3 mg initial dose, 0.1 mg/h supplements) andartificially ventilated with 100% O2 (50 cycles/min, tidal volume:3–4.5 ml). Small adjustments in minute ventilation were made as

*Corresponding author. Tel: �1-503-418-2670; fax: �1-503-418-2501.E-mail address: morrisos@ohsu.edu (S. F. Morrison).Abbreviations: AP, arterial pressure; AP5, D-2-amino-5-phosphonovaler-ate; BAT, brown adipose tissue; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DA, dorsal hypothalamic area; DMH, dorsomedial hypothala-mus; EAA, excitatory amino acid; HR, heart rate; iDMH, dorsomedialhypothalamus ipsilateral; LH, lateral hypothalamic area; MCH, melanin-concentrating hormone; PRV, pseudorabies virus; RPa, raphe pallidus;SNA, sympathetic nerve activity.

Neuroscience 135 (2005) 627–638

0306-4522/05$30.00�0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2005.06.039

627

Neuroscience Letters 383 (2005) 182–187

Changes in EEG activity and hypothalamic temperature as indicesfor non-REM sleep to REM sleep transitions

Paolo Capitania, Matteo Cerrib, Roberto Amicib, Francesca Baracchib, Christine Ann Jonesb,Marco Luppib, Emanuele Perezb, Pier Luigi Parmeggianib, Giovanni Zambonib,∗

a Department of Electronics, Computer Science and Systems, Alma Mater Studiorum-University of Bologna, Italyb 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 2005

Abstract

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 temperature

The change in the behavioural state of non-REM sleep(NREMS) to that of REM sleep (REMS) is a critical period,since physiological regulation shifts from a homeostaticto a non-homeostatic modality[19,21]. We suggest thatthis transition from NREMS to REMS is controlled byintegrative autonomic structures that encompass regulatedchanges occurring in anticipation of the event. This studyis concerned with the changes in two of the physiologicalvariables that may be used to characterize such a transition.

As far as cortical bioelectrical activity is concerned, ithas been shown that this transition does not occur abruptly,but is a smooth change which occurs between the twostates in which some of the features of NREMS graduallylead into REMS. With respect to this, transitions lasting

∗ Corresponding author. Tel.: +39 051 2091742; fax: +39 051 251731.E-mail address:gzamboni@biocfarm.unibo.it (G. Zamboni).

approximately 1 min have been observed in the cat, whereunit firing rate in the posterolateral cortex (lateral andsuprasylvian gyrus) progressively increases from NREMSto REMS[15]. In the rat, cat and mouse, such a transition isusually characterized by specific changes in the electroen-cephalogram (EEG). These consist of successive short bouts(<10 s) of high-amplitude spindles from the anterior cerebralcortex which are associated with a theta rhythm from thedorsal hippocampus[9,10,12]. This distinct pattern of theEEG has lead to the proposal that these brief periods identifya sleep stage which is separate from both NREMS andREMS and has been called either “the intermediate stage ofsleep”[11], “pre-REM sleep”[22] or “transition sleep”[14].

The frequency analysis of the EEG of the rat has shownthat this transition (NREMS to REMS transition, NRT,[4])lasts 30–60 s during which the EEG power density in the Deltaband progressively decreases, whilst that in both the Theta

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.neulet.2005.04.009

SLEEP, Vol. 28, No. 6, 2005

INTRODUCTION

EXPOSURE TO AN AMBIENT TEMPERATURE OUTSIDE THE APPROPRIATE THERMONEUTRAL RANGE CHANG-ES THE AMOUNT AND DISTRIBUTION OF THE DIFFER-ENT stages of the wake-sleep cycle in several different species.1-3 These changes may be a consequence of the differences in the capacity to regulate body temperature across the different stages of the wake-sleep cycle.3,4 In particular, it has been shown that thermoregulatory responses like shivering, panting, and peripher-al vasomotion are suppressed during rapid eye movement (REM) sleep5 due to a change in the activity of the central thermostat at the preoptic-hypothalamic level.6

In early studies carried out in the cat, a short-term exposure to

different low ambient temperatures was shown to primarily af-fect REM-sleep occurrence and that the observed decrease was proportional to the ambient temperature of exposure, while more-complex effects were observed on non-REM (NREM) sleep.1,2 With respect to the latter, “spindle sleep” was progressively de-pressed at ambient temperatures below 0°C, while slow-wave sleep was maximally decreased at an ambient temperature of 5°C but increased toward control levels as the temperature was low-ered.1,2,7 This depression in sleep occurrence by the exposure to low ambient temperature has now been confirmed by many stud-ies on different species.8-15

Exposure to low ambient temperature also clearly influences sleep occurrence when animals are allowed to recover at normal 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 have been confirmed in recent studies on the albino rat, in which it was observed that both REM-sleep loss and the subsequent REM-sleep rebound were quantitatively related to the thermal load (duration of the exposure × decrease in ambient temperature with respect to normal laboratory conditions).17-19 It has also been shown that the amount of NREM sleep is less affected during recovery, since no substantial increase in its amount has been found in either the cat1,2 or the rat.14 However, an increase in the electroencephalo-gram (EEG) power density in the delta band (0.75-4 Hz) during NREM sleep has been observed in the rat.14

Cold Exposure and Sleep in the Rat: Effects on Sleep Architecture and the Electroencephalogram Matteo 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, MD1

1Department 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 Electronics, Computer Science and Systems, Alma Mater Studiorum-University of Bologna, Italy

Study Objectives: Acute exposure to low ambient temperature modifies the wake-sleep cycle due to stage-dependent changes in the capacity to regulate body temperature. This study was carried out to make a sys-tematic analysis of sleep parameters during the exposure to different low ambient temperatures and during the following recoveries at ambient tem-perature 24°C.Design: Electroencephalographic activity, hypothalamic temperature, and motor activity were studied during a 24-hour exposure to ambient temper-atures ranging from 10°C to -10°C and for 4 days during the recovery. Setting: Laboratory of Physiological Regulation during the Wake-Sleep Cycle, Department of Human and General Physiology, Alma Mater Stu-diorum-University of Bologna.Subjects: Twenty-four male albino rats.Interventions: Animals were implanted with electrodes for electroen-cephalographic recording and a thermistor for measuring hypothalamic temperature.Measurements and Results: Wake-sleep stage duration and the electro-encephalographic spectral analysis performed by fast Fourier transform were compared among baseline, exposure, and recovery conditions. The amount of non-rapid eye movement sleep was slightly depressed by cold exposure, but no rebound was observed during the recovery period. Delta

power during non-rapid eye movement sleep was decreased in animals exposed to the lowest ambient temperatures and increased during the first day of the recovery. In contrast, rapid eye movement sleep was greatly depressed by cold exposure and showed an increase during the recovery. Both of these effects were dependent on the ambient temperature of the exposure. Moreover, theta power was increased during rapid eye move-ment sleep in both the exposure and the first day of the recovery.Conclusion: These findings show that sleep-stage duration and electro-encephalogram power are simultaneously affected by cold exposure. The effects on rapid eye movement sleep appear mainly as changes in the duration, whereas those on non-rapid eye movement sleep are shown by changes in delta power. These effects are temperature dependent, and the decrease of both parameters during the exposure is reciprocated by an increase in the subsequent recovery.Key words: Low ambient temperature, sleep deprivation, NREM sleep, REM sleep, single REM sleep, sequential REM sleep, delta power den-sity, theta power densityCitation: Cerri M; Ocampo-Garces A; Amici R et al. Cold exposure and sleep in the rat: effects on sleep architecture and the electroencephalo-gram. SLEEP 2005;28(6):694-705.

Cold Exposure and Sleep in the Rat—Cerri et al

Disclosure StatementThis was not an industry supported study. Drs. Zamboni, Cerri, Ocampo-Garces, Amici, Baracchi, Capitani, Jones, Luppi, Perez, and Parmeggiani have indicated no financial conflicts of interest.

Submitted for publication November 2004Accepted for publication February 2005Address correspondence to: Giovanni Zamboni, MD, Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna,Piazza P.ta S. Donato, 2, I-40126 Bologna, Italy; Tel: 39 051 2091742; Fax: 39 051 251731; E-mail: gzamboni@biocfarm.unibo.it

694

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 2004

Abstract

In the rat the exposure to an ambient temperature (Ta) of �10 8C induces an almost total REM sleep deprivation that results in a

proportional rebound in the following recovery at normal laboratory Ta when the exposure lasts for 24 h, but in a rebound much lower than

expected when the exposure lasts 48 h. The possibility that this may be related to plastic changes in the nervous structures involved in the

control 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 ventromedial

hypothalamic nucleus (VMH) and, as a control, the cerebral cortex (CC). Second messenger concentration was determined in animals either

stimulated by being exposed to hypoxia, a depolarizing condition that induces maximal second messenger accumulation or unstimulated, at

the end of a 24-h and a 48-h exposure to �10 8C and also between 4 h 15 min and 4 h 30 min into recovery (early recovery). At the end of

both 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 low

accumulation in hypoxia. The normal capacity of cAMP accumulation was only restored in the early recovery following 24 h of exposure, but

not following 48 h of exposure, suggesting that this may be a biochemical equivalent of the REM sleep inhibition observed during 48 h of

exposure and which is carried over to the recovery.

D 2004 Elsevier B.V. All rights reserved.

Theme: Endocrine and autonomic regulation

Topic: Osmotic and thermal regulation

Keywords: 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

first applied to the cat and was shown to affect REM sleep

more selectively than NREM sleep [32]. In the rat, it has

been observed that the exposure to low Ta induces a REM

sleep loss and an immediate rebound which is proportional

to the thermal load of exposure (Ta level by duration of

exposure) when animals were returned to recover at normal

laboratory Ta [2,3,4,15]. However, it has been observed that

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.07.002

Abbreviations: cAMP, adenosine 3V:5V-cyclic monophosphate; CC,

cerebral cortex; IP3, d-myo-inositol 1,4,5-trisphosphate; LC, locus coer-

uleus; PO-AH, preoptic-anterior hypothalamic area; Ta, ambient temper-

ature; VMH, ventromedial hypothalamic nucleus

* Corresponding author. Dipartimento di Fisiologia umana e generale

Piazza di Porta San Donato 2, 40127 Bologna BO, Italy. Tel.: +39 051

2091742; fax: +39 051 251731.

E-mail address: gzamboni@biocfarm.unibo.it (G. Zamboni).

Brain Research 1022 (2004) 62–70

www.elsevier.com/locate/brainres