1. 2984 • The Journal of Neuroscience, February 13, 2013 • 33(7):2984 –2993
Behavioral/Cognitive
The Inhibition of Neurons in the Central Nervous Pathways
for Thermoregulatory Cold Defense Induces a Suspended
Animation State in the Rat
Matteo Cerri, Marco Mastrotto,* Domenico Tupone,* Davide Martelli, Marco Luppi, Emanuele Perez,
Giovanni Zamboni, and Roberto Amici
Department 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 it
would avoid the adverse consequence of the powerful autonomic activation evoked by external cooling. Previous attempts to systemically
inhibit metabolism were successful in mice, but practically ineffective in nonhibernators. Here we show that the selective pharmacolog-
ical inhibition of key neurons in the central pathways for thermoregulatory cold defense is sufficient to induce a suspended animation
state, resembling natural torpor, in a nonhibernator. In rats kept at an ambient temperature of 15°C and under continuous darkness, the
prolonged inhibition (6 h) of the rostral ventromedial medulla, a key area of the central nervous pathways for thermoregulatory cold
defense, by means of repeated microinjections (100 nl) of the GABAA agonist muscimol (1 mM), induced the following: (1) a massive
cutaneous vasodilation; (2) drastic drops in deep brain temperature (reaching a nadir of 22.44 Ϯ 0.74°C), heart rate (from 440 Ϯ 13 to
207 Ϯ 12 bpm), and electroencephalography (EEG) power; (3) a modest decrease in mean arterial pressure; and (4) a progressive shift of
the EEG power spectrum toward slow frequencies. After the hypothermic bout, all animals showed a massive increase in NREM sleep
Delta power, similarly to that occurring in natural torpor. No behavioral abnormalities were observed in the days following the treatment.
Our results strengthen the potential role of the CNS in the induction of hibernation/torpor, since CNS-driven changes in organ physiology
have been shown to be sufficient to induce and maintain a suspended animation state.
Introduction esized that two basic players take part in inducing a reduction in
Suspended animation is a temporary and fully reversible condi- metabolism: humoral factors (Andrews, 2007) and the CNS
tion 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 of
factors (Melvin and Andrews, 2009). The cellular and molecular several substances interfering with cell metabolism (Scanlan et
mechanisms 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 approach
abolic 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 and
2008FY7K9S).
*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), a
E-mail: matteo.cerri@unibo.it.
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 heart
land, 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 Mental
Health, 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. Waking and Sleeping following Water Deprivation in the
Rat
Davide 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 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.
unibo.it/PortaleEn/Research/Services+for+teachers+and+researchers/National+Regional+and+Local+Funding/default.htm). 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.it
Introduction 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 suggests
eye movement sleep (REMS) when compared to non rapid eye that the change in hypothalamic integrative activity in this sleep
movement sleep (NREMS) [1]. In particular, during NREMS a stage should concern structures related to thermoregulation, rather
stable 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 the
contrast, during REMS a high variability of the autonomic impairment in physiological regulation during REMS may be
outflow, which leads to large irregularities in arterial blood relevant for the understanding of this sleep state, we sought to test
pressure, heart rate, and respiratory rhythm, is concomitant with the observed independence of osmoregulation from autonomic
an impairment of thermoregulation [1–4]. This impairment has changes in sleep. To this end, REMS occurrence and overall W-S
been considered to be a visible consequence of a change in regulation were assessed in rats during exposure to a three-day
hypothalamic integrative activity, disabling, during REMS, the water deprivation (WD) protocol. This condition is known to
autonomic feedbacks sustaining body homeostasis [1]. According- constitute an osmotic challenge leading to the progressive
ly, Wake-Sleep (W-S) states change when a tonic maintenance of engagement of the whole set of mechanisms maintaining body
the hypothalamic regulation is needed, as it occurs during the fluid homeostasis [8,9]. Moreover, since a REMS rebound was
exposure to a low ambient temperature (Ta). In these conditions, observed during the recovery (R) period following cold exposure
Wake increases, NREMS is variably affected and REMS is always
[5,6,10,11,12,13], W-S assessment was continued for two days
reduced 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 antidiuretic
PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e46116

3. Environ Sci Pollut Res
DOI 10.1007/s11356-012-1266-5
ECOTOXICOLOGY AND ENVIRONMENTAL TOXICOLOGY : NEW CONCEPTS, NEW TOOLS
Effects of chronic exposure to radiofrequency
electromagnetic 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 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). Most
involvement of energy status in vital physiological functions. of the observed effects of RF-EMF exposure were dependent
We 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 body
ulatory processes). Thirteen juvenile male Wistar rats were cooling, whereas energy intake increases. Our results show
exposed to continuous RF-EMF for 5 weeks at 24 °C of air that RF-EMF exposure can induce energy-saving processes
temperature (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, the
functions 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 rat
episodes was greater in the RF-EMF-exposed group, indepen-
dently of Ta (+42.1 % at 24 °C and +31.6 % at 31 °C). The
other effects of RF-EMF exposure on several sleep parameters
Introduction
were dependent on Ta. At 31 °C, RF-EMF-exposed animals
had 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 energy
Responsible editor: Philippe Garrigues balance involves a complicated interaction between energy
A. 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 structures
Jules 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 in
e-mail: nathalie.loos@u-picardie.fr 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-rapid
PériTOX Laboratory (EA 4285-UMI01), VIVA/TOXI,
National Institute of Industrial Environment and Risks (INERIS),
eye movement sleep (NREMS) and rapid eye movement
Parc ALATA BP2, sleep (REMS): the larger the energy intake, the longer the
60550 Verneuil-en-Halatte, France rat will sleep (Danguir and Nicolaidis 1985). As far as
thermal status and feeding behaviour are concerned, body
M. Cerri
Department 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. J. Sleep Res. (2010) 19, 394–399 Osmoregulation and sleep
doi: 10.1111/j.1365-2869.2009.00810.x
Hypothalamic osmoregulation is maintained across the
wake–sleep cycle in the rat
MARCO LUPPI1, DAVIDE MARTELLI1, ROBERTO AMICI1, FRANCESCA
BARACCHI2, MATTEO CERRI1, DANIELA DENTICO1, EMANUELE
P E R E Z 1 and G I O V A N N I Z A M B O N I 1
1
Department 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
The issue of generalizing the impairment of thermoregula-
INTRODUCTION
tion during REMS to all hypothalamic integrative mechanisms
Rapid eye movement sleep (REMS) is characterized by has not been addressed so far from an experimental point of
changes in physiological regulation, which differentiate this view. A way to address this problem is that of studying
sleep 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 intense
has 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 the
been 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 almost
would explain the high degree of instability of the autonomic completely integrated at the hypothalamic level in mammals
activity during REMS (Parmeggiani, 1980, 1988, 1994). (Denton et al., 1996).
In these species, the most efficient defense of osmolality is
Correspondence: Giovanni Zamboni, MD, Department of Human and
General Physiology, Alma Mater Studiorum-University of Bologna,
provided by the release of arginine-vasopressin (AVP), the
Piazza 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: giovanni.zamboni@unibo.it rons of the supraoptic nucleus (SON) and paraventricular
394 Ó 2010 European Sleep Research Society

5. Neuroscience 165 (2010) 984 –995
CUTANEOUS VASODILATION ELICITED BY DISINHIBITION OF THE
CAUDAL PORTION OF THE ROSTRAL VENTROMEDIAL MEDULLA OF
THE FREE-BEHAVING RAT
M. 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 and
Dipartimento di Fisiologia Umana e Generale, Alma Mater Studiorum Morrison, 2003). Antagonism of GABAA receptors in the
Università 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 in
ling 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 leads
rostrally, to the rostral portion of the nucleus of the facial to vasodilation of the tail skin in the anaesthetized rat
nerve (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 mediating
thermoregulatory 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 artery
the 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 the
activity of neurons in the RVMM(io). Microinjection of the RVMM, between the levels corresponding to the rostral
GABAA agonist, muscimol, within either the RVMM(fn) or
portion of nucleus of the facial nerve and the rostral portion
the 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 between
disinhibition of RVMM(io) neurons produced a rapid cutane- the rostral portion of the RVMM, containing more putative
ous vasodilation. We conclude that the tonically-active neu- glutamatergic neurons expressing the vesicular glutamate
rons driving cutaneous vasoconstriction, likely sympathetic transporter, VGLUT3, and the caudal portion of the RVMM,
premotor neurons previously described in the RVMM(fn), are
also located in the RVMM(io). However, in the RVMM(io),
where more serotoninergic neurons have been identified
these 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, it
scious animal and that promotes a cutaneous vasodilation has been proposed that the neural substrate in the RVMM
upon relief of this inhibition. Whether the vasodilator neurons for the control of cutaneous vasomotion is represented by
located in the RVMM(io) play a role in thermoregulation re- a set of VGLUT3-positive, glutamatergic neurons directly
mains to be determined. © 2010 IBRO. Published by Elsevier
projecting to the intermediolateral column (IML) of the
Ltd. All rights reserved.
spinal cord (Nakamura et al., 2004). These data have led
Key words: infrared thermography, thermoregulation, sym- to a theoretical paradigm in which sympathetic outflow to
pathetic nervous system, cutaneous vasomotion, muscimol, cutaneous blood vessels is proportional to the level of
bicuculline 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 now
lations 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), where
Nalivaiko, 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 of
E-mail address: matteo.cerri@unibo.it (M. Cerri).
Abbreviations: AP, arterial pressure; BAT, brown adipose tissue; EKG,
RVMM neurons, within the rostro-caudal section of the
electrocardiogram; HR, heart rate; IML, intermediolateral nucleus of rostral pole of the inferior olive (RVMM(io)), in controlling
the 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 extensive
rostral ventromedial medulla at the level of the rostral inferior olivary
nucleus; 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. European Journal of Neuroscience
European Journal of Neuroscience, Vol. 30, pp. 651–661, 2009 doi:10.1111/j.1460-9568.2009.06848.x
BEHAVIORAL NEUROSCIENCE
c-Fos expression in preoptic nuclei as a marker of sleep
rebound 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 Zamboni1
1
Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Piazza P.ta S. Donato, 2, I-40126
Bologna, Italy
2
Research 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 laboratory
Ta. As two POA subregions, the ventrolateral preoptic nucleus (VLPO) and the median preoptic nucleus (MnPO), have been
proposed 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 in
the early phase of the recovery period. Moreover, the dynamics of the sleep rebound during recovery were studied in a separate
group of animals. The results show that c-Fos expression increased in both the VLPO and the MnPO during cold exposure, but not in
a specific subregion within the VLPO cluster counting grid (VLPO T-cluster). During the recovery, concomitantly with a large rapid eye
movement sleep (REMS) rebound and an increase in delta power during non-rapid eye movement sleep (NREMS), c-Fos expression
was high in both the VLPO and the MnPO and, specifically, in the VLPO T-cluster. In both nuclei, P-CREB expression showed
spontaneous variations in basal conditions. During cold exposure, an increase in expression was observed in the MnPO, but not in
the VLPO, and a decrease was observed in both nuclei during recovery. Dissociation in the changes observed between c-Fos
expression 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 the Exposure to low ambient temperature (Ta) represents a useful tool
regulation of body temperature (Romanovsky, 2007), and has been for inducing physiological sleep deprivation, which is followed by a
clearly recognized as a sleep-promoting site (Szymusiak et al., 2007), sleep rebound after a return to normal laboratory conditions
probably 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 exposure
2003). 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, REMS
occurrence (Szymusiak et al., 2007). It has been proposed that two pressure was shown to increase dramatically, leading to an intense
POA 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 less
processes on the basis of the results of unit recording (Szymusiak challenging for non-rapid eye movement sleep (NREMS), as, under a
et al., 1998; Suntsova et al., 2002, 2007), anatomical tracer (Sherin 24-h exposure to a Ta of )10 °C protocol, the changes in both NREMS
et 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 VLPO
activation is related to sleep occurrence and ⁄ or to an increase in and MnPO in sleep-related processes under a long-term physiological
sleep 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 of
Correspondence: Dr Giovanni Zamboni, as above. )10 °C was shown to dampen the maximum accumulation capacity of
E-mail: giovanni.zamboni@unibo.it
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 the
ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

7. J. Sleep Res. (2008) 17, 166–179 Sleep in animals
doi: 10.1111/j.1365-2869.2008.00658.x
Cold exposure impairs dark-pulse capacity to induce REM sleep
in the albino rat
FRANCESCA 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 1
1
Department 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.
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 of
sleep (REMS), which are usually identified on the basis of the integration and largely depends on the activity of regulatory
level 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 it
from both Wake and NREMS consists of an operational represents a physiological challenge for the organism, partic-
ularly when under unfavourable ambient conditions, such as
Correspondence: Roberto Amici, Department of Human and General
Physiology, 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: roberto.amici@unibo.it been shown not to occur abruptly (Gottesmann, 1996;
166 Ó 2008 European Sleep Research Society

8. REM Sleep
Cold Exposure and Sleep in the Rat: REM Sleep Homeostasis and Body Size
Roberto 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
1
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- Research
Division, 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 seeking
TION AND/OR IN THE INTENSITY OF SLEEP AND THAT a homeostatically regulated sleep parameter have shown that
the occurrence of sleep reduces sleep propensity. The outcome NREMS is substantially regulated in terms of intensity, and
of 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.2,8-11
states, NREM sleep (NREMS) and REM sleep (REMS), which As far as NREMS is concerned, the power density in the
cyclically 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 the
out 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 of
interaction between NREMS and REMS rebounds has been either sleep deprivation or sleep restriction.9,13
observed 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-term
circadian 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.14-17
support from the European Sleep Research Society through a sponsor- The long-term component of REMS regulation is expressed in
ship from Sanofi-Aventis. The other authors have indicated no financial the total amount of REMS during the days following a total
conflicts of interest.
sleep or selective REM sleep deprivation. A precise REMS con-
servation has been observed in both the cat and the rat, since
Submitted for publication July, 2007
the rebound in REMS has been found to be proportional to the
Accepted for publication December, 2007
Address correspondence to: Roberto Amici, MD, Department of Human total loss of REMS during the deprivation period. 11,18-22 This
and General Physiology, Alma Mater Studiorum-University of Bologna, precise quantitative regulation of total REMS amount may not
Piazza P.ta S. Donato, 2, I-40126 Bologna, Italy; Tel: +39-051-2091735; occur following an extended period of sleep deprivation or
Fax: +39-051-2091737; E-mail: roberto.amici@unibo.it sleep restriction.9,13,23 Whereas changes in EEG power are an
SLEEP, Vol. 31, No. 5, 2008 708 REM Sleep Homeostasis and Body Size—Amici et al

9. Available online at www.sciencedirect.com
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 2007
Abstract
The effects of a single intraperitoneal administration of lithium, a drug used to prevent the recurrence of mania in bipolar disorders, were
determined 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 an
hypoxic 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 significant
decrease in the concentration of cAMP in PO-AH at normal laboratory Ta ; (iv) a significant increase of IP3 concentration in CC following exposure
to low Ta . The earliest and most sensitive effects of lithium appear to be those concerning sleep. These changes are concomitant with biochemical
effects that, in spite of a systemic administration of the substance, may be differentiated according to the second messenger involved, the brain
region 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 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 by
insomnia [8], but a consistent alteration of sleep appears to affect shorter exposures, that is, characterized by a smaller REM sleep
the first REM episode, which occurs at a latency shorter than loss [3]. The second set showed that these changes in REM sleep
that 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 accumulate
disturbances [38], it has been observed that the normalization adenosine 3 :5 cyclic monophosphate (cAMP) was significantly
of REM sleep occurrence is concomitant with a symptomatic reduced in the preoptic-anterior hypothalamic area (PO-AH), the
improvement in depressed patients [8]. Likewise, the adminis- thermoregulatory region involved in the control of the negative
tration 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 study
fax: +39 051 2091737. whether the similarity of changes in REM sleep occurrence
E-mail address: christineann.jones@unibo.it (C.A. Jones). caused 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

10. Neuroscience 140 (2006) 711–721
CORTICOTROPIN RELEASING FACTOR INCREASES IN BROWN
ADIPOSE TISSUE THERMOGENESIS AND HEART RATE THROUGH
DORSOMEDIAL HYPOTHALAMUS AND MEDULLARY
RAPHE PALLIDUS
M. CERRIa,b AND S. F. MORRISONa* the raphe pallidus. © 2006 Published by Elsevier Ltd on
a
Neurological Sciences Institute, Oregon Health & Science University, behalf of IBRO.
505 Northwest 185th Avenue, Beaverton, OR 97006, USA
b
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, Italy
Abstract—Corticotropin releasing factor, acting at hypotha-
Corticotropin releasing factor (CRF), identified in 1981
lamic corticotropin releasing factor receptors, contributes to (Vale et al., 1981), is a neuropeptide hormone synthesized
the neural signaling pathways mediating stress-related re- by neurons in the parvocellular paraventricular nucleus
sponses, as well as those involved in maintaining energy (PVN) of the hypothalamus that acts on pituitary cells to
balance homeostasis. Sympathetically-regulated lipid metab- stimulate the secretion of adrenocorticotropic hormone
olism 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 a
ways 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 regulation
ventricle (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 adipose
tissue 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 identify
cleus of the hypothalamus, elicited sustained increases in
brown adipose tissue sympathetic nerve activity, brown adi-
the central sites of action of CRF to stimulate metabolic
pose tissue temperature, expired CO2 and heart rate. These energy consumption have indicated that neurons in the
sympathetic responses to i.c.v. corticotropin releasing factor preoptic anterior hypothalamus (POA) are involved in the
were 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 food
site 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 regulating
hypothalamus. The increases in brown adipose tissue sym- BAT sympathetic outflow and BAT thermogenesis and
pathetic outflow, brown adipose tissue temperature and heart energy expenditure (Morrison, 2004b) has increased since
rate 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 contains
ticotropin releasing factor release within the preoptic area
activates a sympathoexcitatory pathway to brown adipose putative BAT sympathetic premotor neurons (Bamshad et
tissue 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 dorsomedial
includes 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: morrisos@ohsu.edu (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), by
sure; 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 hypothalamus
releasing 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 of
arterial 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 BAT
0306-4522/06$30.00ϩ0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2006.02.027
711

11. Neuroscience 135 (2005) 627– 638
ACTIVATION OF LATERAL HYPOTHALAMIC NEURONS STIMULATES
BROWN ADIPOSE TISSUE THERMOGENESIS
M. CERRIa,b AND S. F. MORRISONa* trating hormone (MCH)-containing neurons reside within
a
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 feeding
b
Dipartimento di Fisiologia Umana e Generale, Universita’ degli Studi occurs (Sakurai, 2002, 2005; Sakurai et al., 1998) and in
di 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 in
Abstract—The lateral hypothalamic area, containing orexin
neurons, 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 a
mine if activation of lateral hypothalamic area neurons influ- hypermetabolic phenotype with a lower body fat mass
ences 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 ((Keesey
chloralose-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 in
icant 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 latter
sure (؉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 see
nists into dorsomedial hypothalamus promptly reversed the
(Park et al., 1988)). Since these responses could have
increases in brown adipose tissue sympathetic nerve activ-
ity, brown adipose tissue temperature and heart rate evoked
arisen either from interruption of axons coursing through
by disinhibition of neurons in lateral hypothalamic area. We the lesion site or from eliminating the activity of neurons in
conclude that neurons in the lateral hypothalamic area can LH, questions remain concerning the role of LH neurons in
influence brown adipose tissue sympathetic nerve activity, the regulation of energy expenditure in BAT. The present
brown adipose tissue thermogenesis and heart rate through study was initiated to determine the effects of cell-specific
pathways that are dependent on the activation of neurons in activation of LH neurons on the sympathetically-regulated
dorsomedial hypothalamus and raphe pallidus. © 2005 IBRO. thermogenesis of BAT.
Published by Elsevier Ltd. All rights reserved.
Key words: raphe pallidus, dorsomedial hypothalamus, bicu- EXPERIMENTAL PROCEDURES
culline, orexin, sympathetic nerve activity, thermoregulation.
General procedures
Experiments were performed in accordance with the National
Neurons in the lateral hypothalamus (LH) and perifornical Institutes of Health Guide for the Care and Use of Laboratory
region have been implicated in the hypothalamic regulation Animals (NIH publication no. 80 –23, 1996) and under protocols
of energy homeostasis (Kalra et al., 1999), including both approved by the Institutional Animal Care and Use Committee of
feeding and energy metabolism which, in turn, determine Oregon Health and Science University. The experimental design
and anesthetic protocol minimized the number of animals used
the level of stored energy and body weight. Populations of and prevented any indication of pain perception. Sprague–Dawley
LH 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. Heart
E-mail address: morrisos@ohsu.edu (S. F. Morrison). rate (HR) was derived from the AP signal. After the animals were
Abbreviations: AP, arterial pressure; AP5, D-2-amino-5-phosphonovaler- positioned prone in a stereotaxic frame according to the approach
ate; 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 and
mus; EAA, excitatory amino acid; HR, heart rate; iDMH, dorsomedial with a spinal clamp on the T10 vertebra, they were paralyzed with
hypothalamus ipsilateral; LH, lateral hypothalamic area; MCH, melanin- D-tubocurarine (0.3 mg initial dose, 0.1 mg/h supplements) and
concentrating 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 as
0306-4522/05$30.00ϩ0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2005.06.039
627

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 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 been
proposed: the NREMS to REMS transition indicator value, NIV [J.H. Benington et al., Sleep 17 (1994) 28–36]. However, such transitions
are not always followed by a REMS episode, but are often followed by an awakening. In the present study, the relationship between changes
in EEG activity and hypothalamic temperature (Thy), taken as an index of autonomic activity, was studied within a window consisting of the
60 s which precedes a state change from a consolidated NREMS episode. Furthermore, the probability that a transition would lead to REMS
or wake was analysed. The results showed that, within this time window, both a modified NIV (NIV60 ) and the difference between Thy at the
limits of the window (ThyD ) were related to the probability of REMS onset. Both the relationship between the indices and the probability of
REMS onset was sigmoid, the latter of which saturated at a probability level around 50–60%. The efficacy for the prediction of successful
transitions from NREMS to REMS found using ThyD as an index supports the view that such a transition is a dynamic process where the
physiological 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 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 and
since physiological regulation shifts from a homeostatic suprasylvian gyrus) progressively increases from NREMS
to a non-homeostatic modality [19,21]. We suggest that to REMS [15]. In the rat, cat and mouse, such a transition is
this 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 bouts
changes occurring in anticipation of the event. This study (<10 s) of high-amplitude spindles from the anterior cerebral
is concerned with the changes in two of the physiological cortex which are associated with a theta rhythm from the
variables 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 identify
has been shown that this transition does not occur abruptly, a sleep stage which is separate from both NREMS and
but is a smooth change which occurs between the two REMS and has been called either “the intermediate stage of
states 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: gzamboni@biocfarm.unibo.it (G. Zamboni). band 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

13. 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
Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Italy; 2Programa de Fisiología y Biofísica, Instituto de
1
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 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. Delta
INTRODUCTION different low ambient temperatures was shown to primarily af-
fect REM-sleep occurrence and that the observed decrease was
EXPOSURE 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.1,2
ES 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-wave
These changes may be a consequence of the differences in the sleep was maximally decreased at an ambient temperature of 5°C
capacity 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 to
thermoregulatory 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.8-15
sleep5 due to a change in the activity of the central thermostat at Exposure to low ambient temperature also clearly influences
the 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 have
Disclosure Statement been confirmed in recent studies on the albino rat, in which it was
This was not an industry supported study. Drs. Zamboni, Cerri, Ocampo-
observed that both REM-sleep loss and the subsequent REM-sleep
Garces, Amici, Baracchi, Capitani, Jones, Luppi, Perez, and Parmeggiani
rebound were quantitatively related to the thermal load (duration
have indicated no financial conflicts of interest.
of the exposure × decrease in ambient temperature with respect
Submitted for publication November 2004 to normal laboratory conditions).17-19 It has also been shown that
Accepted for publication February 2005 the amount of NREM sleep is less affected during recovery, since
Address correspondence to: Giovanni Zamboni, MD, Department of Human no substantial increase in its amount has been found in either the
and 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) during
251731; E-mail: gzamboni@biocfarm.unibo.it NREM sleep has been observed in the rat.14
SLEEP, Vol. 28, No. 6, 2005 694 Cold Exposure and Sleep in the Rat—Cerri et al

14. Brain Research 1022 (2004) 62 – 70
www.elsevier.com/locate/brainres
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 À10 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-cyclic
monophosphate (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 À10 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 IP3
concentration 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
Abbreviations: cAMP, adenosine 3V -cyclic monophosphate; CC,
:5V first applied to the cat and was shown to affect REM sleep
cerebral cortex; IP3, d-myo-inositol 1,4,5-trisphosphate; LC, locus coer- more selectively than NREM sleep [32]. In the rat, it has
uleus; PO-AH, preoptic-anterior hypothalamic area; Ta, ambient temper- been observed that the exposure to low Ta induces a REM
ature; VMH, ventromedial hypothalamic nucleus
sleep loss and an immediate rebound which is proportional
* Corresponding author. Dipartimento di Fisiologia umana e generale
Piazza di Porta San Donato 2, 40127 Bologna BO, Italy. Tel.: +39 051 to the thermal load of exposure (Ta level by duration of
2091742; fax: +39 051 251731. exposure) when animals were returned to recover at normal
E-mail address: gzamboni@biocfarm.unibo.it (G. Zamboni). 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