InstitutNationaldelaSante ´etdelaRechercheMe ´dicaleU628,Universite ´Claude-Bernard-LyonI,69373LyonCedex08,France
nuclei of the posterior hypothalamus. They are all characterized by triphasic broad action potentials. They are active only during
irregular firing pattern. Their activity varies in the different waking states, being lowest during quiet waking, moderate during active
of sleep (drowsy state), and remain silent during slow-wave sleep and paradoxical (or rapid eye movement) sleep. They exhibit a
induction of wakefulness per se, but in the maintenance of the high level of vigilance necessary for cognitive processes. Conversely,
Since von Economo’s anatomopathological observations on the
viral encephalitic epidemic of 1918, several lines of experimental
amus in the maintenance of wakefulness (W) (Moruzzi, 1972).
Experimental destruction or inactivation of posterior hypotha-
lamic neurons results in somnolence and hypersomnia. In addi-
els of insomnia, produced either by destruction of the preoptic
para-chlorophenylalanine, a 5-hydroxytryptamine (serotonin or
5-HT) synthesis inhibitor, amphetamine, a psychostimulant, or
Of the posterior hypothalamic neurons, the histaminergic neu-
neuronal histamine (HA) in the mammalian brain and are con-
tained in widely branching neuronal pathways influencing large
target fields, such as the noradrenergic neurons in the nucleus
locus ceruleus and serotonergic neurons in the dorsal raphe nu-
cleus (DRN) (Schwartz et al., 1991; Wada et al., 1991; Haas and
Panula, 2003). The HA system is therefore thought to play an
important role in a wide variety of physiological functions, in-
cluding sleep/waking regulation, learning, memory, attention,
and control of affective states (Schwartz et al., 1991; Wada et al.,
1991; Haas and Panula, 2003). HA neurons play an important
of HA neurons in the rat posterior hypothalamus (Haas and
Reiner, 1988; Greene et al., 1990; Haas, 1992; Llinas and Alonso,
mation is available about the unit activity of identified HA neu-
rons in vivo in general and across wake–sleep states in rodents,
including the mouse in particular, despite its increasing use in
experimental models. Determination of the activity of HA neu-
rons across sleep–waking cycles is a prerequisite for an under-
standing of their precise roles in behavioral state control and of
their physiological functions. Here we report for the first time
that the unit activity of identified mouse HA neurons is waking
specific and related to the high level of vigilance necessary for
Animals and surgery. All experimental procedures followed European
Economic Community Guidelines (86/609/EEC) and the Policy on Eth-
ics approved by the Society for Neuroscience (1993). Every effort was
made to minimize the number of animals used as well as any pain and
Male adult C57BL/6 mice (28–35 g at the time of surgery; Harlan,
Gannat, France) were used. Under pentobarbital anesthesia (50 mg/kg
This study was supported by Institut National de la Sante ´ et de la Recherche Me ´dicale U628, Claude Bernard
CorrespondenceshouldbeaddressedtoKazuyaSakai,InstitutNationaldelaSante ´etdelaRechercheMe ´dicale
U628, Universite ´ Claude-Bernard-Lyon 1, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France. E-mail:
10292 • TheJournalofNeuroscience,October4,2006 • 26(40):10292–10298
i.p.), electrodes were implanted to record neocortical and hippocampal
electroencephalograms (EEGs), neck muscle activity [electromyogram
tip) was implanted in the dorsal hippocampus. For EMG and EKG re-
the skull using dental acrylic cement so that the cranium could be pain-
holder (SA-8; Narishige, Tokyo, Japan). A small hole was drilled in the
skull above the posterior hypothalamus and covered by antibiotic cream
for the subsequent insertion of microelectrodes. After a recovery period
being placed on a cotton sheet inside a plastic box, painlessly restraining
the head with a semichronic head holder and preventing large body
movements with a cotton-coated plastic covering. During experiments,
the head was covered to reduce visual stimuli. Under these conditions,
wave sleep (SWS), and paradoxical [or rapid eye movement (REM)]
sleep (PS) during all experiments lasting 6–8 h. During experiments,
sugared water was regularly given through a fine tube attached to a
Extracellular single-unit and polygraphic recordings. Single neuronal
activity was recorded extracellularly using a glass pipette microelectrode
filled with either a 0.5 M sodium acetate solution containing 2% pon-
Laboratories, Burlingame, CA). The electrode was vertically inserted in
increments of 1–3 ?m using a pulse motor microdrive manipulator
(MO-81; Narishige, Tokyo, Japan). The neuronal activity was amplified
and filtered (NeuroLog; Digitimer, Hertfordshire, UK) with a cutoff fre-
quency of 100 Hz and then digitized at a sampling rate of 16.7–20.0 kHz
using a CED 1410 data processor (Cambridge Electronic Design, Cam-
bridge, UK). The polygraphic signals were also digitized at a sampling
rate of either 504 or 512 Hz and stored on a personal computer.
At the end of each experiment, pontamine sky blue was injected from
as to mark one or two recording sites in each electrode track.
Juxtacellular labeling with neurobiotin. Nb was dissolved at a concen-
the sleep–waking cycle, Nb was delivered through the recording elec-
intensity (?10 nA) under continuous electrophysiological monitoring
taneous firing without damaging the recorded neuron. The position of
the microelectrode was also adjusted by moving the electrode with the
hydraulic manipulator to prevent obvious cellular damage. Within 8 h
after the end of Nb labeling, the mice received a lethal dose of pentobar-
bital and were processed for histochemistry.
Histochemistry. At the end of experiments, the animals were deeply
anesthetized with pentobarbital and then perfused through the ascend-
ing aorta with 50 ml of Ringer’s solution, followed by 150 ml of fixative
consisting of 4% N-(3-dimethylaminopropyl)-carbodiimide (CD;
Sigma-Aldrich, Saint Quentin, France) in 0.1 M phosphate buffer (PB),
removed and postfixed for 48 h at 4°C in PB containing 4% CD and 1%
PF and placed in PB containing 30% sucrose for 48 h at 4°C. Twenty
micrometer sections were then cut on a cryostat and stored in 0.1 M PBS
containing 0.3% Triton X-100 (PBST) and 0.1% sodium azide until
For juxtacellular Nb application experiments, the sections were first
incubated overnight at 4°C with ABC (diluted 1:2000; Vector Laborato-
ries) and then, after three rinses, were processed for visualization using
DAB (Vector Laboratories) as chromogen. The sections were then incu-
bated for 7–8 d at 4°C with rabbit anti-histamine antibodies (Millipore,
Billerica, MA), diluted 1:80,000 in PBST. After several washes, the sec-
tions were incubated overnight at 4°C with biotinylated anti-rabbit IgG
antibodies (Vector Laboratories), diluted 1:1000 in PBST. After several
rinses, they were incubated for 90 min at room temperature with ABC
(diluted 1:2000; Vector Laboratories) and then processed for visualiza-
tion using DAB–nickel (Vector Laboratories) as chromogen.
Data analysis. Sleep–waking stages were defined using the EEG and
defined as the first 3 s period from the onset of EEG synchronization
during the transition from W to SWS. SWS was defined by sustained
high-voltage slow waves in the EEG and lowered EMG activity. PS was
defined by sustained theta waves and decreased ? waves in the EEG and
using a Fast Fourier Transform routine and the Spike2 analysis program
(Cambridge Electronic Design). The lead from dorsal hippocampus–
? (6.0–12.0 Hz) frequency bands. Analysis of unitary activity was per-
2–10 s bins for each of the following states: (1) attentive waking (AtW),
elicited by touching the tail with a soft brush or removing the cover
a low-amplitude (desynchronized) EEG with a sustained EMG activity;
movements, including grooming (2–10 s bin); (3) quiet waking (QW),
variation for the firing interval (SD/mean discharge interval) was calcu-
lated during the waking states. Statistical analysis was performed using
one-way ANOVA or a two-tailed t test, a p value of ?0.05 or 0.01 being
considered, respectively, as significant or highly significant.
the TM region of the posterior hypothalamus of 17 mice during
the complete sleep–wake cycle, including at least one episode of
42 neurons recorded in the TM (Figs. 1, right side, 2a). In the rat
brain, the TM has been subdivided into either three (medial,
ventral, and diffuse) (Ericson et al., 1987) or five (E1–E5) (Ina-
gaki et al., 1988) subgroups. As shown in Figure 1 (left side), the
HA neurons were also found in these regions in the mouse pos-
terior hypothalamus. In the mouse TM, we found a strikingly
homogeneous group of neurons that were clearly distinguished
from other posterior hypothalamic neurons in terms of spike
to be HA neurons on the basis of their characteristic action po-
tential waveform, their spontaneous discharge pattern across the
sleep–waking cycle, and their double immunostaining with jux-
tacellularly applied Nb and HA, as described below.
All HA TM neurons displayed a broad, triphasic action potential
(Fig. 3A). The HA TM neurons could be clearly distinguished in
terms of wave form from other posterior hypothalamic neurons,
which exhibited either narrow (Fig. 3B1) or broad (Fig. 3B2)
biphasic action potentials, such as those of “waking-active” neu-
rons (see below).
All HA TM neurons discharged tonically during W and fell
completely silent during both SWS and PS (Fig. 4) and are here-
after referred to as “waking-specific” neurons. During waking
displayed a slow (?10 Hz) tonic, repetitive, irregular firing pat-
Takahashietal.•HistaminergicNeuronalActivityintheMouseJ.Neurosci.,October4,2006 • 26(40):10292–10298 • 10293
rized in supplemental Table 1 (available at www.jneurosci.org as
different waking states, being lowest during QW without motor
behavior (Mean ? SD, 3.00 ? 1.21 spikes/s), moderate during
AW with motor activities (4.12 ? 1.42 spikes/s), and highest
during AtW (5.15 ? 1.99 spikes/s) elicited by touching the tail
with a soft brush or removal of the cover placed over the mouse.
The difference in firing rate for the three states was statistically
in discharge rate was also seen between AtW and AW and be-
tween AW and QW ( p ? 0.05, two-tailed t test).
In addition to the 42 waking-specific HA TM neurons, we
found 12 waking-active TM neurons, which discharged at a
and SWS, and at the lowest rate during PS (Fig. 1, indicated by
circles on the right side, and Fig. 6A,B). The discharge rate and
firing pattern of these waking-active neurons were variable, and
differing from the HA TM waking-specific neurons. Their mean
5.09, 2.76 ? 1.57, 0.86 ? 1.04, and 0.30 ? 0.25 Hz, respectively.
neurons during behavioral state transitions. During the transi-
tion from W to SWS (Fig. 7A, t2), they stopped discharging be-
(range, 450.2–3016.0). During the transition from SWS to W
(Fig. 7A, t1), they displayed firing not before, but after, the onset
of EEG desynchronization, with a mean latency of 800.18 ? 62.7
animals entered a steady sleep cycle consisting of multiple SWS
and PS episodes, brief interruptions of sleep caused by phasic
body movements were not accompanied by any spike discharge
(Fig. 4, arrowheads on the hypnogram). During the transition
from PS to W (Fig. 7A, t3), they exhibited unit discharges soon
after the end of the PS episode, defined by the interruption of
sustained theta waves and the onset of EEG desynchronization
from the end of PS was 3849.2 ? 833.8 ms (range,
When applied during SWS, an arousing sound stimulus (hand
tion to desynchronization (Fig. 7B). HA TM neurons either re-
or did not respond at all (latency, ?2 s), especially when the
and Fig. 7B, 2). The mean minimal latency (? SEM) for waking-
active neurons was 105.3 ? 26.6 ms (range, 14.1–313.1), signifi-
cantly shorter ( p ? 0.01, two-tailed t test) than that of waking-
Successful recordings across all sleep–wake states were made
from 28 neurons, which were then labeled with Nb and found to
be located in the TM region of the posterior hypothalamus (Fig.
1, right side). Eight were HA immunopositive (Fig. 2b), and all
eight were waking-specific neurons, characterized by a triphasic
broad action potential. The remaining 20 neurons were HA im-
munonegative (Fig. 2c), and all were characterized by a biphasic
were SWS/PS-active, and 7 were state indifferent (Fig. 1).
The present study is the first to investigate single-unit discharge
of HA TM neurons in nonanesthetized, head-restrained mice
across wake–sleep states. We have identified, for the first time in
and found that they are active only during W and that their
millary nucleus; VTM, ventral tuberomammillary nucleus; f, fornix; MM medial mammillary
nucleus; LM, lateral mammillary nucleus; mt, mammillothalamic tract; MTu, medial tuberal
nucleus; PH, posterior hypothalamic area; pm, principal mammillary tract; PMV, ventral pre-
10294 • J.Neurosci.,October4,2006 • 26(40):10292–10298Takahashietal.•HistaminergicNeuronalActivityintheMouse
activity is related to a high level of vigilance. They cease firing
animals are not fully alert and respond with a long delay, or do
not respond at all, to an arousing stimulus if it does not elicit an
overt alert state. Several lines of evidence support the view that
these waking-specific neurons are all HA. They were recorded in
rons are found, but not in other regions of
the posterior hypothalamus. These mouse
TM neurons exhibited a characteristic
spike shape and a remarkably homoge-
neous pattern of unit activity in different
behavioral states and in response to an
arousing stimulus, as discussed below.
Double immunostaining with Nb and HA
showed that the waking-specific neurons
characterized by a triphasic broad action
a narrow or broad biphasic action poten-
tial were HA negative.
Characteristics of HA TM neurons in vitro and in vivo
All mouse TM waking-specific neurons had a broad triphasic
action potential. This finding is consistent with those previously
obtained by in vitro intracellular recording from identified HA
TM neurons in the rat brain (Haas and Reiner, 1988; Greene et
al., 1990; Kamondi and Reiner, 1991; Llinas and Alonso, 1992;
Reiner and Kamondi, 1994; Stevens et al., 2001).
to fire spontaneously at a slow-regular rate (0.5–10 Hz) at the
resting membrane potential (Stevens et al., 2001). In urethane-
anesthetized rats, HA TM neurons are reported to discharge
slowly, with a relatively regular pattern (Reiner and McGeer,
1987). In the present in vivo experiments in nonanesthetized,
head-restrained mice, HA TM neurons were completely quies-
cent during both the drowsy state and sleep, and, during waking
states, displayed a tonic (? 10 Hz) but irregular firing pattern, as
shown by the large coefficient of variation for the spike interval
(CV ? 0.5), suggesting that mouse HA TM neurons are very
silent only during deep SWS and PS (Vanni-Mercier et al., 1985;
Sakai et al., 1990; John et al., 2004). Steininger et al. (1999) re-
cently reported the presence of PS (REM)-off neurons in the rat
the TM. These neurons appear to be similar to the waking-active
neurons described in the present study but differ from HA TM
shape, and reduced but persistent discharge during SWS and PS.
In the present study, two waking-active neurons were labeled
with Nb, and both were found to be HA negative.
Three HA receptors, H1, H2, and H3, have been identified in the
tic actions, whereas H3receptors, located on histaminergic and
other cell somata, dendrites, and axons, cause autoinhibition of
HA TM neurons and inhibition of the release of other neuro-
transmitters (Haas and Panula, 2003). H1receptors mediate ex-
tion of H1 receptor antagonists, commonly known as
antihistamines, evokes sedation in humans (Nicholson, 1983;
Schwartz et al., 1991). Our previous study showed that local in-
jection of muscimol in the TM causes sleep (Lin et al., 1989),
suggesting a role of HA TM neurons in the sedation and sleep.
waking-specific neurons are characterized by a broad triphasic action potential, whereas
peak (D1), the first zero crossing (D2), the peak of the afterhyperpolarization (D3), and the
Takahashietal.•HistaminergicNeuronalActivityintheMouseJ.Neurosci.,October4,2006 • 26(40):10292–10298 • 10295
are reported to play a role in the mechanisms of wake–sleep reg-
ulation and in the pathophysiology of narcolepsy/cataplexy
(Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000). Orx/
Hcrt directly excites HA TM neurons in vitro in rat brain slice
preparations (Bayer et al., 2001; Eriksson et al., 2001; Yamanaka
et al., 2002). Recent in vivo studies in mice showed that intrace-
system (Hong et al., 2005) and causes a significant increase in W
in wild-type mice but not in H1receptor gene knock-out mice
is mainly mediated by activation of the HA system.
In the present experiments in mice, all HA TM neurons fell
silent during quiet waking before the onset of EEG synchroniza-
tion observed during the drowsy state, suggesting that the cessa-
tion of activity of HA TM neurons may play a role in the induc-
tion of SWS. The mechanisms underlying this cessation of
activity during sleep are not known, but it is currently believed
that it is caused by GABAergic inputs from sleep-active neurons
in the preoptic and basal forebrain areas (Saper et al., 2001).
firing of HA TM neurons. It has been shown in vitro (Haas and
Reiner, 1988; Greene et al., 1990; Llinas and Alonso, 1992;
Stevens et al., 2001) that, when depolarizing pulses are applied
from a hyperpolarized membrane potential level, there is a long
delay before activation of cells because of a pronounced voltage-
gated transient outward current (A-type current) (Connor and
Stevens, 1971). It is possible that mouse HA TM neurons are
hyperpolarized during sleep and that the transient outward cur-
rent ensures that the membrane potential remains below thresh-
the end of PS, as shown by the interruption of theta waves and the onset of EEG desyn-
chronization. The small arrows with an “s” indicate short awakenings elicited by the
10296 • J.Neurosci.,October4,2006 • 26(40):10292–10298 Takahashietal.•HistaminergicNeuronalActivityintheMouse
old during sleep. Cessation of activity of HA TM neurons may
therefore play a role in the maintenance of sleep.
As shown in the present study, HA TM neurons display
arousal-specific neuronal activity. Although the activity of
human HA TM neurons remains unknown, presumed HA TM
specific neuronal activity similar to that seen in mice (Vanni-
Mercier et al., 1985; Sakai et al., 1990; John et al., 2004). An
increase in histaminergic transmission enhances W, whereas its
blockade causes somnolence (for review, see Lin, 2000). HA in-
duces a switch in neuronal firing mode from rhythmic burst to
single spike activity in thalamic relay neurons (McCormick and
Williamson, 1991) and an increase in spiking by reducing spike-
frequency adaptation (McCormick and Williamson, 1989). In
addition, HA stimulates cholinergic neurons of the brainstem
and basal forebrain (Khateb et al., 1995; Lin et al., 1996; Crochet
rons of the DRN (Sakai and Crochet, 2000; Brown et al., 2002),
both implicated in waking and attention. Collectively, these data
indicate an important role of HA in the control of arousal.
drowsy state, characterized by a low vigilance level, remained
were not fully alert, and responded with a long delay, or dis not
respond at all, to an arousing stimulus if it did not elicit an overt
alert state. These findings suggest that the activity of HA TM
HA neuronal activity results in somnolence. These data are in
good agreement with those of our recent study showing that KO
mice for histidine decarboxylase, the HA-synthesizing enzyme,
display a deficit in W and signs of somnolence when faced with a
novel environment (Parmentier et al., 2002). Additional detailed
studies in freely behaving animals are required to determine
whether or not a particular waking state drives the neuronal ac-
tivity of HA TM neurons or vice versa. The alternation between
waking and sleep is determined by multiple arousal- and sleep-
promoting systems, which are widely distributed in the brain
(Jones, 2005). Precise knowledge of the unit activity profiles of
these systems during the state transition should be helpful to
understand how each system contributes to the generation
and/or maintenance of the behavioral states.
In conclusion, the present study shows, for the first time in
of vigilance necessary for cognitive processes. Conversely, cessa-
tion of their activity may play an important role in both the
initiation and maintenance of sleep.
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