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The Journal of Neuroscience, September 1, 2002, 22(17):7695-7711
Anatomical, Physiological, and Pharmacological Characteristics of
Histidine Decarboxylase Knock-Out Mice: Evidence for the Role of Brain
Histamine in Behavioral and Sleep-Wake Control
Régis
Parmentier1,
Hiroshi
Ohtsu2,
Zahia
Djebbara-Hannas1,
Jean-Louis
Valatx1,
Takehiko
Watanabe2, and
Jian-Sheng
Lin1
1 Institut National de la Santé et de la
Recherche Médicale U480, Department of Experimental Medicine,
Faculty of Medicine, Claude Bernard University, 69373 Lyon, France, and
2 Department of Cellular Pharmacology, Tohoku University,
School of Medicine, Aoba-ku, Sendai 980-8575, Japan
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ABSTRACT |
The hypothesis that histaminergic neurons are involved in brain
arousal is supported by many studies. However, the effects of the
selective long-term abolition of histaminergic neurons on the
sleep-wake cycle, indispensable in determining their functions, remain
unknown. We have compared brain histamine(HA)-immunoreactivity and
the cortical-EEG and sleep-wake cycle under baseline conditions or
after behavioral or pharmacological stimuli in wild-type (WT) and
knock-out mice lacking the histidine decarboxylase gene (HDC /). HDC/mice showed an increase in paradoxical sleep, a decrease in cortical EEG power in  -rhythm during waking (W), and a decreased EEG slow wave sleep/W power ratio. Although no major difference was
noted in the daily amount of spontaneous W, HDC/mice showed a
deficit of W at lights-off and signs of somnolence, as demonstrated by
a decreased sleep latencies after various behavioral stimuli, e.g.,
WT-mice placed in a new environment remained highly awake for 2-3 hr,
whereas HDC/mice fell asleep after a few minutes. These
effects are likely to be attributable to lack of HDC and thus of HA. In WT mice, indeed, intraperitoneal injection of
 -fluoromethylhistidine (HDC-inhibitor) caused a decrease in W,
whereas injection of ciproxifan (HA-H3 receptor antagonist) elicited W. Both injections had no effect in HDC/mice. Moreover,
PCR and immunohistochemistry confirmed the absence of the HDC gene and
brain HA-immunoreactive neurons in the HDC/mice. These data
indicate that disruption of HA-synthesis causes permanent changes in
the cortical-EEG and sleep-wake cycle and that, at moments when high
vigilance is required (lights off, environmental change... ), mice
lacking brain HA are unable to remain awake, a prerequisite condition
for responding to behavioral and cognitive challenges. We suggest that
histaminergic neurons also play a key role in maintaining the brain in
an awake state faced with behavioral challenges.
Key words:
wakefulness; sleep-wake cycle; cortical activation; arousal; histaminergic neurons; histamine; histidine decarboxylase
knock-out mice; cortical EEG; behavioral challenge
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INTRODUCTION |
Wakefulness (W) and cortical
arousal, defined on the electroencephalogram as low-voltage fast
cortical activity, are classically explained by a generally accepted
concept, i.e., the reticular ascending activating system initially
proposed by Moruzzi and Magoun (1949) . According to this, the main
ascending excitatory sources originate from neuronal elements within
the brainstem reticular formation and transit in the intrathalamic and
extrathalamic relays, finally activating the entire cortex by
widespread intrathalamocortical and extrathalamocortical systems
(Moruzzi, 1972). The importance of the intrathalamocortical system is
supported by much recent experimental data (Steriade, 1991), whereas
other studies have identified several subcortical structures that serve
as the extrathalamocortical systems in cortical activation. Among such
systems, the basal forebrain has received special attention (Steriade
and Buzsaki, 1990; Jones, 2000). In addition, classical and recent
studies support a critical role of the posterior hypothalamus and
histaminergic neurons in cortical activation and W.
Histaminergic perikarya are found in the tuberomammillary nucleus (TMn)
and adjacent areas of the posterior hypothalamus (Panula et al., 1984;
Steinbusch et al., 1984; Watanabe et al., 1984; Lin et al., 1986a), a
heterogeneous area containing multiple neuronal populations and known
to be important in waking mechanisms, because its lesions cause
hypersomnia (Moruzzi, 1972; Sakai et al., 1990; Lin, 2000).
Histaminergic cells send widespread projections to most cerebral
regions, including those known to be important in sleep-wake control,
such as the cortex, thalamus, and posterior and preoptic/anterior
hypothalamus, and to the forebrain and brainstem aminergic and
cholinergic structures (Inagaki et al., 1988; Panula et al., 1989; Lin
et al., 1993, 1996). In these target areas, histamine(HA) modulates
neuronal activity-excitability via H1, H2, H3, and, possibly,
unidentified receptors (Schwartz et al., 1991; Haas, 1992; Brown et
al., 2001). In freely moving animals, presumed histaminergic neurons
discharge tonically and specifically during W (Vanni-Mercier et al.,
1984; Sakai et al.,1990; Steininger et al., 1999). Administration of
various substances impairing histaminergic transmission increases slow
wave sleep (SWS), whereas enhancement of transmission promotes W (Lin
et al., 1988; Monti et al., 1991). Muscimol-induced inactivation of the
posterior hypothalamus containing HA cells results in hypersomnia in
both normal or experimentally induced insomniac cats (Lin, 2000).
Finally, inhibition of HA synthesis in the same area increases SWS,
whereas inhibition of HA degradation elicits long-lasting arousal (Lin et al., 1986b, 1988).
These results support the potential importance of histaminergic neurons
in arousal, but the effects of selective long-term abolition of HA
neurons on the sleep-wake cycle, indispensable in determining their
role, remain unknown. Although attempts have been made in cats to
abolish neuronal activity in the posterior-hypothalamus either
temporarily (Lin et al., 1989) or permanently (Sallanon et al., 1988),
these experimental approaches affect not only histaminergic cells, but
also nonhistaminergic neurons codistributed in the same or adjacent
regions, and so the observed hypersomnia cannot be definitively
attributed to the loss of HA cells. Among the nonhistaminergic cells
present in the posterior-hypothalamus, a subpopulation, located in the
perifornical area, just rostrodorsal to the histaminergic TMn and
containing orexin-hypocretin neuropeptides has recently been
identified in the mammalian CNS (de Lecea et al., 1998; Sakurai et al.,
1998). There is evidence that orexin deficiency might be responsible
for the pathogenesis of human and animal narcolepsy (Lin et al., 1999)
and that, like HA neurons, orexin-containing cells may be involved in
arousal by their widespread projections. It is therefore necessary to
clarify the respective role of histaminergic and orexin neurons in
sleep-wake control using animal models with selective abolition of
each of these systems. In terms of the orexin system, the sleep-wake
cycle has been studied in knock-out (KO) (Chemelli et al., 1999) and
genetic abolition (Hara et al., 2001) models. As to the HA system,
Ohtsu et al. (2001) have recently developed KO mice lacking histidine decarboxylase (HDC), the sole enzyme responsible for HA synthesis, and
have demonstrated the absence of HA synthesis in these HDC/mice, thus providing a unique experimental model for determining the impact
of long-term selective abolition of the HA system on the sleep-wake
cycle. The present study was therefore designed to compare the brain HA
system, cortical EEG, and sleep-wake cycle under spontaneous
conditions or after behavioral or pharmacological stimuli in wild-type
and HDC/ mice.
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MATERIALS AND METHODS |
Animals and surgery
Fifteen pairs of male inbred (129/Sv strain) wild-type (HDC+/+
or WT) and histidine decarboxylase gene knock-out (HDC/ or KO) mice
were used. The HDC/ mice were generated according to previously
described procedures (Ohtsu et al., 2001). Briefly, linearized
targeting construct was electroporated into R1 embryonic stem cells,
derived from 129/Sv embryo (Nagy et al., 1993). The chimeric mice,
generated with the confirmed embryonic stem cells, were crossed with
129/Sv mice to obtain the inbred +/ mice. The animals were kept on
129/Sv genomic background. All experiments followed EEC Directive
(86/609/EEC), and every effort was made to minimize the number of
animals used and any pain and discomfort.
At the age of 12 weeks and with a body weight of 27-33 gm, the animals
were chronically implanted, under deep sodium pentobarbital anesthesia
(60-70 mg/kg, i.p.), with six cortical electrodes (gold-plated tinned
copper wire,  = 0.4 mm; Filotex, Draveil, France) and three
muscle electrodes (fluorocarbon-coated gold-plated stainless steel
wire, = 0.03 mm; Cooner Wire, Chatworth, CA) to record the
electroencephalogram (EEG) and electromyogram (EMG) and to monitor the
sleep-wake cycle. All electrodes were previously soldered to a
multichannel electrical connector, and each was separately insulated
with a covering of heat-shrinkable polyolefin-polyester tubing. The
cortical electrodes were inserted into the dura through three pairs of
holes ( = 0.5 mm) made in the skull, located, respectively, in
the frontal (1 mm lateral and anterior to the bregma), parietal (1 mm
lateral to the midline at the midpoint between the bregma and lambda),
and occipital (2 mm lateral to the midline and 1 mm anterior to the
lambda) cortices. The muscle electrodes were inserted into the neck
muscles. Finally, the electrode assembly was anchored and fixed to the
skull with Super-Bond (Sun Medical Co., Shiga, Japan) and dental
cement. This implantation allowed stable polygraphic recordings to be
made for >4 months.
Polygraphic recording and data acquisition and analysis
After surgery, the animals were housed individually in
transparent barrels ( 20 cm, height 30 cm) in an insulated
sound-proofed recording room maintained at an ambient temperature of
22 ± 1°C and on a 12 hr light/dark cycle (lights on at 7:00
A.M.), food and water being available ad libitum. In some
animals and for some experiments, a video camera with infrared and
digital time recording capabilities was set up in the recording room to
observe and score the animal's behavior during both the light and dark phases. After a 3-4 d recovery period, the mice were habituated to the
recording cable for 10 d before starting polygraphic recordings.
Cortical EEG (ipsilateral and contralateral frontoparietal and
fronto-occipital leads) and EMG signals were amplified, digitized with
a resolution of 200 and 100 Hz, respectively, and computed on a CED
1401 Plus (Cambridge, UK). Using a Spike2 script and with the
assistance of spectral analysis using the fast Fourier transform,
polygraphic records were visually scored by 30 sec epochs for
wakefulness (W), slow wave sleep (SWS), and paradoxical sleep (PS)
according to previously described criteria validated for mice (Valatx,
1971; Valatx and Bugat, 1974).
To avoid any variation caused by the positioning of cortical
electrodes, the cortical EEG used for power spectral density analysis
was captured from frontoparietal leads, set with reference to the
bregma, lambda, and midline in all mice. EEG power spectra were
computed for consecutive 30 sec epochs within the frequency range of
0.8-60 Hz using a fast Fourier transform routine. The data were
collapsed in 0.4 Hz bins. On the basis of visual and spectral analysis,
epochs containing artifacts occurring during active waking (with large
movements) were visually identified and omitted from the spectral
analysis when the threshold value in the 0-1 Hz band was exceeded;
this represented 1.96 ± 0.94% of the total recording time. The
power densities obtained for each state were summed over the frequency
band of 0.8-60 Hz (total power). To standardize the data, all power
spectral densities at the different frequency ranges, i.e.,  ,
0.8-2.4 Hz; , 3-9 Hz; , (spindle) 9-19 Hz;  , 20-30 Hz;
 , 30-60 Hz; and +, 20-60 Hz, were expressed as a percentage
relative to the total power (e.g., power in the range/power in the
0.8-60 Hz range) of the same epochs. To evaluate contrast in the
cortical EEG between SWS and W or PS, we used an EEG power ratio
determined by the averaged cortical EEG total power density during SWS
divided by that during either W or PS.
Experimental procedures
In each experiment, recordings were simultaneously made from an
equal number (usually in batches of three) of HDC/ and HDC+/+ mice.
The mice were submitted to the following experimental procedures.
Spontaneous cortical EEG and sleep-wake cycle.
During the period of days 15-45 after surgery, drug-naive mice (11 pairs) were subjected to three separate 24 hr recording sessions,
beginning at 7:00 P.M. During each recording session, the animals were
left undisturbed. The data from the two sets of mice were then compared.
Cortical EEG and sleep-wake cycle after behavioral stimuli.
Recordings were made from HDC/ and HDC+/+ mice for 24 hr after each
of the three tests described below, which were performed in a random
sequence. As a criterion of sedation and drowsiness, the latencies to
SWS and PS, defined as the time between the end of the stimuli and the
onset of the first SWS or PS episode lasting >30 sec, were also
measured. The three tests consisted of: (1) a simulation of injection
(at either 10:00 A.M. or 8:00 P.M.; n = 26 from 13 pairs of animals), consisting of the handling of the animal and sham
intraperitoneal injection without needle insertion; (2) a change of
litter (at 2:00 P.M.; n = 36 from 15 pairs of mice),
which was a routine care performed at light phase every 4-6 d to clean
the cage and which usually causes a period of waking and behavioral
excitation in rodents; in this test, we compared the excitability of
the two groups of mice following this routine care; (3) a new
environment, the mice being transferred for 4 hr from their habitual
transparent barrel to an opaque rectangular box (21 × 30 cm,
height 20 cm, with open field); in this test, the ability of the two
genotypes to remain awake after this environmental change was tested.
Each mouse was subjected to this test four times separated by an
interval of 10-14 d, twice at 2:00 P.M. when the animals normally
being sleepy for ~80% of the time (defined as "sleepy period";
n = 22 from 11 pairs of mice) (Fig.
1A), and twice at 6:00
P.M. when they would normally be awake a majority of the time (defined
as "awake period"; n = 18 from nine pairs of mice)
(Fig. 1A). Sleep-wake stages during their stay in
the new environment were compared between the two groups and with the
baseline recordings for the same group.
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Figure 1.
Quantitative comparison of spontaneous sleep-wake
parameters in inbred HDC+/+ and HDC/ mice. HDC+/+ mice,
filled symbols and columns; HDC/
mice, unfilled symbols and columns.
A, Mean hourly values (± SE) of the sleep-wake states.
The gray area corresponds to the period between 6:00 and
10:00 P.M., and the total sleep-wake values for each state during this
period for both genotypes are indicated in the histogram.
B, Means (± SE) of sleep-wake stages for the 12 hr
light (Day) and dark (Night) periods and
the 24 hr period. C, Mean values (± SE) of episode
duration and number of episodes of each sleep-wake stage for all 24 hr
recordings. Note that, compared with HDC +/+ mice, HDC / mice
exhibit the following: (1) a deficit of W immediately before and after
lights-off (A), without major change in the daily
amount of W or SWS (B); (2) an increase in PS,
mainly in the light phase, because of an increase in the number of
episodes (A-C); and (3) a fragmented sleep-wake
architecture, with shortened episode duration and increased number of
episodes in W and SWS (C). Note also the small
interindividual SDs for the sleep-wake stages within each genotype
group, indicating that each group was genetically homogenous
(n = 33, corresponding to 3 × 24 hr
recordings for 11 animals of each genotype). *p < 0.05; **p < 0.01; ***p < 0.001; ****p <0.0001, using a two-tailed
t test after significance in a two-way ANOVA for
repeated measures).
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Cortical EEG and the sleep-wake cycle after drug
administration. To compare the effects of drugs acting on the
histaminergic system, the two genotypes of mice were injected
intraperitoneally with the following agents with an interval of at
least 7 d between injections, subsequent recordings being made for
24 hr: (1) saline alone (0.1 ml) and saline containing
(S)--fluoromethylhistidine (-FMH; 50 mg/kg;
Merck, Sharp, & Dohme; n = 14 for each genotype; seven
pairs of mice), a specific HDC inhibitor (Kollonitsch et al., 1978;
Garbarg et al., 1980; Maeyama et al., 1982); injections were given at
4:00 P.M. (2) Saline alone (0.1 ml) and saline containing cyclopropyl-(4-(3-(1H-imidazol-4-yl) propyloxy) phenyl) ketone (ciproxifan, 1 mg/kg; Bioprojet, Paris, France) (n = 9 for each genotype; nine pairs of mice), a potent and specific
antagonist of histamine-H3 receptor (Ligneau et al., 1998) that
controls histamine release and synthesis by autoinhibition; injections were given at 10:00 A.M. (sleepy period). In addition, some animals (nine pairs) were injected subcutaneously with either saline, alone or
containing histamine dihydrochloride (HA; Sigma, St. Louis, MO; 1 mg/kg, dissolved in saline and adjusted to pH 7) during either the
light (10:00 A.M.; n = 9) or dark phase (8:00 P.M.;
n = 9) to assess the effect of peripheral HA on the
cortical EEG and the sleep-waking cycle. All drugs, expressed as salt
weight, were dissolved immediately before use. Results in the saline- and drug-injected animals were compared.
Detection of the HDC gene using the PCR.
To confirm the genotypes of the mice used with respect to the HDC gene,
at the end of the experiments, tail biopsies were taken from all mice
and analyzed by PCR. The WT allele was amplified using primers located within the HDC gene fragment that was replaced by the neomycin resistant gene in the KO mice. These primers were 5'-AGT GAG GGA CTG
TGG CTC CAC GTC GAT GCT-3' (complementary to HDC gene 833-862) and
5'-TAC AGT CAA AGT GTA CCA TCA TCC ACT TGG-3' (HDC gene 980-951), the
expected product size being 147 base pairs. The mutant allele was
amplified using primers located within the
neor gene, these being 5'-AAA CAT CGC ATC
GAG CGA GCA CGT ACT CGG-3' and 5'-ATG TCC TGA TAG CGG TCC GCC ACA CCC
AGC-3', with an expected product size of 244 base pairs. These two sets
of primers were included concomitantly and PCR was performed using 40 cycles of 30 sec at 94°C, 1 min at 64°C, and 1 min at 72°C,
followed by one cycle at 72°C for 10 min. The whole reaction mix was
then fractionated on a 2% agarose gel, and the PCR product was
visualized by ethidium bromide staining.
Histamine immunohistochemistry. To examine the fate of brain
histaminergic neurons after HDC gene disruption, at the end of the
other experiments, HA immunohistochemistry was performed as described
by Panula et al. (1988), Lin et al. (1993, 1996), and Eriksson et al.
(2001). Briefly, the WT and KO animals were anesthetized with an
overdose of sodium pentobarbital (>100 mg/kg) and perfused transcardially with 50 ml of Ringer's lactate solution containing 0.1% heparin, followed by 60 ml of ice-cold 0.1 M phosphate buffer (PB), pH 7.4, containing 4%
1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide and 0.1%
N-hydroxysuccinimide (both from Sigma). After 48 hr
postfixation in the same solution and 48 hr rinsing in PB containing
30% sucrose and 0.1% sodium azide, the brains were coronally
sectioned (25 µm thickness) on a freezing cryostat. Free-floating
sections were then incubated at 4°C for 84-96 hr on an agitator with
rabbit anti-HA antibody (Delichon, Masala, Finland) diluted
1:20,000-80,000 in PB saline containing 0.3% Triton X-100 (PBS-T) and
0.1% sodium azide. The specificity of the anti-HA antibody has been
demonstrated in several species in previous studies (Panula et al.,
1988; Lin et al., 1993, 1996), including mice (Airaksinen et al.,
1992). After rinses, the sections were submitted to one of the
following procedures:
(1) Some were incubated at 4°C overnight on an agitator with
CyTM3-conjugated anti-rabbit IgG (1: 800; Jackson
ImmunoResearch, West Grove, PA) in PBS-T, then immediately mounted on
glass slides, and coverslipped with glycerin and examined on a light
microscope equipped with epifluorescent illumination with a filter to
view CyTM3. A positive reaction was seen as gold-orange
fluorescent staining of the labeled structures (see Figs. 11,12).
(2) Others were incubated with either anti-rabbit IgG antibody
(1:1000-2000; Jackson ImmunoResearch, West Grove, PA) or biotinylated anti-rabbit IgG antibody (1: 1000-2000; Vector Laboratories,
Burlingame, CA), then, after several rinses, were incubated with rabbit
peroxidase-anti-peroxidase (PAP; 1:2000-40,000; Jackson
ImmunoResearch) or horseradish peroxidase-conjugated streptavidin
(1:40,000; Jackson ImmunoResearch), or a Vectastain ABC kit (1:2000;
Vector Laboratories). Both incubations were in PBS-T at
4°C overnight on an agitator. The sections were then immersed for
5-10 min at room temperature in 0.05 M Tris-HCl
buffer, pH 7.6, containing 0.02% 3,3'diaminobenzidine-4HCl, 0.6%
nickel ammonium sulfate, and 0.003%
H2O2. A positive reaction
resulted in blue-black staining of the labeled structures (somata,
dendrites, axons, and varicosities). Some sections were counterstained
with neutral red to identify topographic and cellular structures.
Finally, all sections were immediately mounted on gelatin-coated glass slides, dried, and coverslipped with Depex for light microscopy.
Whereas the biotin-conjugated reagents proved to be incompatible with
the carbodiimide-perfused mouse tissue, specific clear labeling was
seen using fluorescent staining or nonconjugated IgG/PAP, and the data
presented are those using these methods. The atlas of Franklin and
Paxinos (1996) was used for the anatomical nomenclature of cerebral
regions and for their abbreviations.
Statistical analysis
ANOVA and the post hoc Student's t test
(two-tailed) were used to evaluate differences between HDC+/+ and
HDC/mice in the cortical EEG and sleep-wake parameters under
normal conditions or after treatment and differences in these
parameters between control data (baseline recordings or saline
injection) and data after treatment in the same group of animals; in
the latter case, individual animal served as its own control.
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RESULTS |
General observations
As wild-type (WT or HDC+/+) mice, HDC gene disrupted (HDC/ or
KO) mice appeared to develop normally. No apparent troubles were noted
in terms of fertility, general morphology, movement, or other behaviors
under basal conditions. Nevertheless, compared with HDC+/+ animals,
HDC/ mice seemed to be less reactive when handled, and, at the age
of ~12 weeks, their body weight was greater (31.6 ± 0.9 gm at
the age of 97 ± 7 d; n = 15 vs 27.8 ± 0.5 gm at the age of 93 ± 3 d; n = 15;
p < 0.001; Student's t test). This
difference in body weight increased with age (e.g., the respective weights at 42 weeks were 40.5 ± 2.6 gm; n = 11 vs
33.0 ± 1.1 gm; n = 11; p < 0.02;
Student's t test).
Spontaneous sleep-wake cycle in HDC/ mice
Under basal conditions in which the animals were left undisturbed,
both sets of mice exhibited a circadian sleep-waking rhythm characteristic of 129/Sv (Huber et al., 2000) and other mice (Franken et al., 1999), i.e., with larger amounts of W during the dark period
than during the light one (Fig. 1A,B). Quantitative
analysis of the time spent in each sleep-wake stage during the diurnal and nocturnal periods or over 24 hr showed a significantly greater amount of PS in HDC/mice. This increase occurred mainly during the
light phase because of an increased number of PS episodes and also led
to a PS augmentation of >23% over 24 hr (Fig. 1B), PS episode duration remaining unchanged during both light/dark phases
(Fig. 1C). No direct onset of PS phases from W was seen in
either the lights-on or lights-off period. With respect to W and SWS,
HDC/ mice displayed a significant decrease in episode duration and
an increase in episode number for both states (Fig. 1C) and
during both light/dark phases, e.g., the mean W episode duration over
24 hr was 3.7 min ± 0.1 instead of 4.6 min ± 0.2 seen in
HDC+/+ mice. However, these changes did not result in a significant
change in the total amount of W and SWS during either the light or dark
phase or over 24 hr (Fig. 1B).
Despite this lack of a major change in the daily amount of spontaneous
W, hourly analysis of sleep-wake states (Fig. 1A)
revealed a significantly smaller amount of W in HDC/ mice during
the period of 6:00-10:00 P.M, corresponding to the periods before and
after lights-off at 7:00 P.M. Thus, like most rodents, the HDC+/+ mice
anticipated and responded to lights-off with a significant increase in
W (as a result of increased W episode duration: 6.5 min ± 0.3 vs
4.6 min ± 0.2; the mean value over 24 hr; p < 0.001), accompanied by a high level of behavioral activity, whereas
this characteristic phenomenon was markedly reduced or almost absent in
the HDC/ mice (4.1 ± 0.1 vs 3.7 min ± 0.1, the mean
value over 24 hr; p > 0.05) (see Fig.
1A and examples of polygraphic recordings in Fig.
2A,B). Concomitant to
the deficit in W in HDC/ mice (122.6 ± 2.8 vs 151.2 ± 3.3 min in HDC+/+ mice; p < 0.001) there was an
increase in both SWS and PS (Fig. 1A, boxed areas). The W deficit during this period was compensated for over 24 hr, because there was no major change in the daily total W (Fig.
1B).
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Figure 2.
Typical examples of polygraphic recordings and
corresponding hypnograms illustrating the spontaneous sleep-wake cycle
before and after lights-off and sleep-wake state transitions in HDC+/+
and HDC/ mice. Samples on the hour range (A, B) or
second range (a, b) scale from an HDC+/+ (A,
a) or HDC/ (B, b) mouse, showing the
following: (1) the cortical EEG signs of both genotypes (a,
b); and (2) the decreased waking (A, B) around
the lights-off and reduced cortical electroencephalogram (EEG) SWS/W
amplitude ratio (A, B, a, b) in the HDC/ mouse.
Calibration: 200 µV, 1 sec. EMG, Electromyogram.
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Because an age-related increase in body weight was found in HDC/
mice and to determine the possible effect this might have on the
sleep-wake cycle or vice versa, we also examined the relationship between body weight and sleep-wake parameters in all mice. No correlation was found between body weight (at age of 12 weeks) and the
daily amount of sleep-wake stages in either genotype, except for a
negative correlation with PS (linear regression, p = 0.034) in the HDC/group. At the age of  40 weeks, the body weight
of HDC/ mice increased significantly, whereas their daily amount of
PS remained unchanged (data not shown). These data indicate that there
is no simple causal link between body weight and the sleep-wake change
seen in HDC/ mice. Further studies, such as controlling the
animal's food intake, activity, and metabolism, are required to
determine the mechanisms by which HDC/ mice regulate their body weight.
Characteristics of cortical EEG in HDC/ mice
From the frontoparietal leads (Fig. 2; see Fig. 4) as the
fronto-occipital ones (data not shown), the cortical EEG of both sets
of animals manifested marked and specific changes across the behavioral
states and signs characteristic of mice, i.e., with dominant presence
of frequencies, notably during PS and W. Nevertheless, compared
with HDC+/+ ones, HDC/mice showed changes in the following
aspects:
A reduced cortical EEG SWS/W power ratio
As shown in Figure 2 (see also Figs. 5, 8), one remarkable and
visually detectable change in the cortical EEG in HDC/ mice was a
reduction in the EEG SWS/W amplitude ratio. This observation is
confirmed by an analysis of the averaged cortical EEG SWS/W power
(0.8-60 Hz) (Fig. 3A) showing
that, in HDC+/+ mice, the power ratio was higher during the darkness
than during the light phase, with the maximal ratio found around
lights-off (4:00-10:00 P.M.) (Fig. 3B), and that the ratio
in HDC/ mice was significantly lower than that in HDC+/+ mice,
during either phase or over 24 hr (Fig. 3A). This decreased
ratio was seen during all recorded baseline periods (i.e., days 15-45
after surgery). The reduced SWS/W power ratio was mainly attributable
to a reduced peak power and amplitude of the cortical EEG of SWS.
Although these parameters also decreased during W, that could not
contribute to such a reduced ratio (Fig.
4). In addition, the cortical EEG SWS/PS
power ratio was also lower in HDC/ mice (data not shown). Because
of these changes in the cortical EEG desynchronization/synchronization power ratios in HDC/ mice, we then analyzed each cortical EEG frequency during all sleep-wake states in both HDC+/+ and HDC/ mice. As shown in Figure 4, A1 and A2, both
genotypes displayed state-dependent cortical EEG spectral profiles,
with peak powers of W, SWS, and PS similar to those reported for the
129/Sv (Huber et al., 2000), 129/Ola, or DBA/2J (Franken et al., 1998)
strains. When the power spectral density in each 0.4 Hz bin/total power (0.8-60 Hz) of each 30 sec epoch was compared between the genotypes (Fig. 4B1-B4), we found further changes in
the HDC/ mice:
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Figure 3.
Cortical EEG SWS/W power ratio (0.8-60 Hz) in
HDC+/+ and HDC/ mice during the night or day periods or over 24 hr
of spontaneous recordings or during hours 3-7 after injection of
saline or -FMH. Filled columns, HDC+/+ mice;
unfilled columns, HDC/ mice. A,
Values of spontaneous recordings (n = 11);
B, values of recordings between 7:00 and 11:00 P.M.
(n = 14) after injection of saline or -FMH (50 mg/kg, i.p.) at 4:00 P.M. Note the significantly reduced ratio in
HDC/ mice during normal conditions (A) or
after saline injection (B), and the lack of a
significant difference between HDC+/+ and HDC/ mice in the ratio
after injection of -FMH (B)
(*p < 0.05, °p > 0.05;
two-tailed t test).
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Figure 4.
Mean spectral distribution of cortical EEG power
density in spontaneous sleep-wake states in inbred HDC+/+ and HDC/
mice. The data were obtained from 14 pairs of animals by pooling
consecutive 30 sec epochs during the period of 7:00-10:00 P.M. using
the fast Fourier transform routine within the frequency range of
0.8-60 Hz. A1, A2, Mean absolute power values (in
square microvolts) in each 0.4 Hz frequency bin. Note the
state-dependent profiles of cortical EEG spectra across wakefulness
(W), slow wave sleep (SWS),
and paradoxical sleep (PS) in HDC+/+ (A1)
and HDC/ (A2) mice. B1-B3, Mean
percentage power density calculated as the mean power (in square
microvolts) in each 0.4 Hz frequency bin divided by the total power
(0.8-60 Hz) in the same epoch. The spectra from HDC+/+ mice were set
to the same apparent sizes to those of the same animals in
A1 to facilitate comparison. The inset on
B1 is enlarged view for 0.8-2.4 Hz. B4,
EEG power spectra in HDC/ mice (columns,
n = 14) expressed as a mean percentage change (± SE) relative to those (± SE) in HDC+/+ mice (baseline 0;
n = 14). Note that the HDC/ mice show an
increase in power density of cortical frequency (0.8-2.4 Hz)
during W, a deficit of power density of cortical slow rhythm (3-9
Hz) during W and SWS, and an increase in power density of cortical fast
rhythms (+, 20-60 Hz) during SWS (*p < 0.05; **p <0.01; ***p <0.001;
two-tailed t test).
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An increased power density of cortical activity in range
during W
This increase, limited to the slow range (0.8-2.4 Hz), was
seen only during W (Fig. 4B1, B4).
A decrease in the power density of cortical rhythm
(3-9 Hz)
This was most marked during W, less prominent during SWS, and not
significant during PS (Fig. 4B1-B4). This
deficit of rhythm appears to contribute largely to the decrease in
the peak power and amplitude of the cortical EEG of W and SWS (Fig.
4).
An increase in the power density of cortical fast rhythm ( and
ranges, 20-60 Hz) during SWS (Fig. 4B2,B4)
A smaller increase was also seen during PS and W, but did not
reach statistical significance. No major change was seen in power
density in the spindle or frequencies (including the 9-19 Hz
range) during any state (Fig. 4B1-B4).
Effects of behavioral stimuli on sleep latencies and the
sleep-wake cycle
Consistent with the observation that HDC/ mice were less
reactive when handled, three behavioral tests confirmed an objective sedative behavior in these mice.
Recordings of sleep-wake parameters after a routine litter change
during the light phase or after a behavioral challenge, such as a
simulation of injection, during either the light or dark phase showed
significant shorter latencies to SWS and PS in HDC/ mice than in
HDC+/+ mice (Table 1). There were
however, no major differences between the two genotypes in the
sleep-wake cycle after sleep onset (data not shown).
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Table 1.
Mean latencies (± SE) to slow wave sleep (SWS) and
paradoxical sleep (PS) in HDC+/+ and HDC/ mice after behavioral
challenge or drug administration
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A greater difference in the sleep-wake cycle was seen between the two
genotypes in the new environment test, which consisted of transferring
the mice from their habitual home cage (a transparent barrel) to a new
cage (an opaque rectangular box with open
field). Thus, as shown in Figures 5 and 6
and Table 1, the transfer of the HDC+/+ mice to the new cage elicited
an increase in W over the whole 4 hr period that the mice were in the
new cage, including a significant increase in the latencies to SWS and
PS (Table 1) and an increase in W and decrease in SWS after sleep onset
during their stay in the new cage (Figs. 5, 6). They appeared to be
interested by the new environment, because video recording showed
several exploratory behaviors, such as ambulation around the new cage and rearing. In contrast, HDC/ mice seemed indifferent and
unresponsive, because they fell asleep soon after the environmental
change (Fig. 5, Table 1), and there was no change in W and SWS during
their 4 hr stay in the new cage, compared with their own control data during basal conditions (Fig. 6). This difference in responsiveness to
the new environment was seen both at 2:00 P.M. (sleepy period; Fig. 5, top panels, Table 1), and at 6:00 P.M. (awake
period; Fig. 5, bottom panels, Table 1). Both sets of mice
showed a decrease of ~4-8 min in PS in the new environment compared
with their baseline recordings (Fig. 6) in which they were undisturbed,
presumably as a result of handling. Interestingly, when the animals
were placed back to their home cages after the 4 hr stay, no any
significant difference in term of sleep latencies was noted between the
two genotypes (Table 1), suggesting that novelty plays an important role in the new environment-elicited awakening in the HDC+/+ mice. It
should be mentioned that the object recognition test showed that the
vision of both phenotypes was intact and that the mice appeared not to
be stressed during their stay in the new cage, because a similar degree
of water and food intake and grooming occurred as in the home cage. No
immobility or hyperactivity or other apparently unusual behavioral
signs were seen, suggesting that the animals did not suffer stress or
anxiety in the new environment.
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Figure 5.
Typical examples of polygraphic recordings and
corresponding hypnograms illustrating the effects of an environmental
change on HDC+/+ and HDC/ mice. The environmental change (indicated
by an arrow) consisted of moving the animals from their
habitual transparent barrel cages to opaque rectangular cages at either
2:00 P.M. (sleepy period; top panels) or 6:00 P.M.
(awake period; bottom panels). Note that the HDC +/+
mouse remained awake for >2 hr in the new environment, whereas the
HDC/ mouse fell asleep soon after the test.
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Figure 6.
Quantitative variations of sleep-wake states in
HDC+/+ and HDC/ mice after an environmental change. Top
histograms (A, B), Mean values (± SE in
minutes) of each sleep-wake stage during the 4 hr in which the animals
were in the new environment compared with the baseline recordings for
the same group. Bottom histograms (a, b),
Sleep-wake changes (in minutes) relative to the baseline value for the
same group. Left histograms (A, a),
Environmental change at 2:00 P.M.; right histograms
(B, b), change at 6:00 P.M. Note the significant
increase in waking (W) and decrease in
slow wave sleep (SWS) in HDC+/+ mice compared either
with their own baseline values (A, B) or the values
(bottom, a, b) for HDC/ mice (n = 18 at 2:00 P.M. and 22 at 6:00 P.M. from 9 and 11 pairs of animals).
PS, Paradoxical sleep; °p > 0.05;
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-tailed t test after significance in a two-way ANOVA
for repeated measures.
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Effects of drug administration on the cortical EEG and
sleep-wake cycle
-FMH
Before and after lights-off, W and locomotion increased in the
HDC+/+ mice, whereas these effects were markedly less evident in
HDC/ mice. When the animals were injected intraperitoneally with
-FMH (specific HDC inhibitor, 50 mg/kg,) at 4:00 P.M. (3 hr before
lights-off, in view of the latency of drug action shown in Garbarg et
al., 1980; Maeyama et al., 1982), a progressively developing
significant decrease in W and increase in both SWS and PS were seen in
HDC+/+ mice (Fig. 7, left
plots), results similar to the reduction in W seen in untreated
HDC/ mice during this period under basal conditions. The increase
in SWS and PS was caused by a prolongation of episode duration (data
not shown). In contrast, -FMH injection had no effect on either W,
SWS, or PS in HDC/ mice, (Fig. 7, right plots). The
latency to SWS after injection was not significantly different between
the two genotypes, whereas that to PS was significantly shorter in
HDC/ mice, similar to that seen with a simulation of injection
(Table 1). It can be mentioned here that -FMH at a smaller dose (20 mg/kg) in cats (Lin et al., 1988) and rats (Monti et al., 1988) results
in a similar increase in SWS to that seen here in HDC+/+ mice, but the
effect is not accompanied by an increase in PS; whereas a delayed
(latency >16 hr) increase in PS is seen in rats using a larger dose
(100 mg/kg; Kiyono et al., 1984). It remains to determine whether the
previous negative results on PS were attributable to incomplete
inhibition of the HA synthesis.
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Figure 7.
Effects of -FMH on sleep-wake states in HDC+/+
and HDC/ mice. The curves show the mean hourly
cumulative values (±SE) during the 7 hr after injection of -FMH (50 mg/kg, i.p., at 4:00 P.M.) (filled symbols) or
saline (unfilled symbols). Note the progressive decrease
in wakefulness (W) and increase in both
slow wave sleep (SWS) and paradoxical sleep
(PS) in the HDC+/+ mice (left traces) but
not the HDC/ mice (right traces)
(n = 14 from 7 pairs of mice;
*p < 0.05, **p < 0.01;
two-tailed t test after significance in two-way ANOVA
for repeated measures).
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Concomitant with the behavioral activation before and after lights-off
in HDC+/+ mice, the cortical EEG SWS/W power ratio was highest during
this period (Fig. 3B). As in other circadian periods, the
cortical-EEG SWS/W power ratio in HDC/ mice during this period was
significant lower than that in HDC+/+ mice. -FMH injection had no
effect on this ratio in HDC/ mice, but reduced it in HDC+/+ mice,
with the result that, after injection, there was no longer significant
difference between the two genotypes in the cortical EEG SWS/W power
ratio (Fig. 3B).
Ciproxifan
In HDC+/+ mice, intraperitoneal injection of ciproxifan (1 mg/kg),
a potent and specific HA-H3 receptor antagonist (Ligneau et al., 1998)
at the light phase (10:00 A.M.) caused suppression of cortical slow
activity ( + ranges) and spindles (8-14 Hz), resulting in a
state of total cortical activation (Fig.
8), i.e., low-voltage electrical activity
with dominant waves in the and bands (20-60 Hz). Furthermore,
ciproxifan injection increased the power density of these neocortical
fast rhythms (Fig. 8). The effects on the cortical EEG were manifested
on polygraphic scoring as an almost total waking state, characterized
by a significantly delayed sleep latency (Table 1) and suppression of
SWS and PS, followed by an increase in W after sleep onset, lasting >4
hr (Figs. 8, 9).
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Figure 8.
Effects of ciproxifan on cortical EEG
and sleep-wake states in HDC+/+ and HDC/ mice. Top
traces, HDC+/+ mice; bottom traces, HDC/
mice. Examples of polygraphic recordings, cortical EEG power density
(in square microvolts) in different frequency bands, and the
corresponding hypnograms illustrating suppression of cortical EEG power
at 0.8-8 ( + ) and 8-14 () Hz, marked enhancement of
cortical fast rhythm (+, 30-60 Hz), and a waking state induced
by injection of ciproxifan (1 mg/kg, i.p, at 10:00 A.M., indicated by
the arrow) in an HDC+/+ mouse but not in an HDC/
mouse.
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Figure 9.
Quantitative variations in sleep-wake states in
HDC+/+ and HDC/ mice after ciproxifan injection. Top
histogram, Mean values (± SE, in minutes) of each
sleep-wake stage after injection of saline (control) or ciproxifan (1 mg/kg, i.p., at 10:00 A.M.). Bottom histograms,
Sleep-wake changes (in minutes) relative to the baseline value for the
same group. Note the significant increase in waking
(W) and decrease in slow wave sleep
(SWS) in HDC+/+ mice compared with either their own
baseline values or those of HDC/ mice (n = 9;
for both). *p < 0.05; ***p < 0.001; ****p < 0.0001, two-tailed t
test).
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In HDC/ mice, the same injection of ciproxifan had no
significant effect on either the cortical EEG or the sleep-wake states compared with saline injection of the same animals (Figs. 8, 9).
Histamine dihydrochloride
To assess and compare the influence of peripheral HA, which does
not pass through the blood-brain barrier (Schwartz et al., 1991), on
the cortical EEG and sleep-wake cycle in HDC+/+ and HDC/ mice, HA
was injected subcutaneously during the dark (8:00 P.M.;
n = 9) or light phase (10:00 A.M.; n = 9). HA at a dose of 1 mg/kg had no significant effect on either the
cortical EEG or the sleep-wake states in either genotype compared with
saline injection (n = 9 at either 10:00 A.M. or 8:00
P.M.) of the same animals (data not shown).
Genotype confirmation by PCR
At the end of the experiments, to confirm the HDC genotypes of the
mice, we performed PCR on genomic DNA from tail biopsies from all 15 mice in each group. As shown in Figure
10, a strong HDC signal, corresponding
to a 147 base pair band, was detected in the HDC+/+ mice (animals
16-30 in Fig. 10), and a strong Neor
signal, corresponding to a 244 base pair band, was detected in the
HDC/ mice (animals 1-15), proof that, in the KO animals, the HDC
gene had indeed been disrupted and the
Neor gene inserted.
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Figure 10.
PCR confirmation of genotypes. Lanes
1-15, HDC/ mice; lanes 16-30, HDC+/+ mice.
Note that all HDC+/+ mice displayed a 147 bp band corresponding to the
HDC gene fragment, whereas all HDC /mice showed a 244 bp band
corresponding to the Neor gene fragment.
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Histology and histamine immunohistochemistry
Because the HA system in the mouse brain has not been fully
described in the literature, at the end of the experiments, we performed HA immunohistochemistry and examined the distribution of
histaminergic cell bodies and fibers throughout the brain in both
genotypes to determine the fate of brain histaminergic neurons after
HDC gene disruption.
HDC+/+ mice
Similar to results in the cat (Lin et al., 1986a, 1993), rat
(Panula et al., 1984; Steinbusch and Mulder; 1984; Watanabe et al.,
1984), guinea pig (Airaksinen and Panula, 1988), and mouse (Airaksinen
et al., 1992), in the HDC+/+ mice, HA-immunoreactive neurons were
located almost exclusively in the posterior hypothalamus; a few
appeared more rostrally, restricted to a region dorsal to the optic
tract in the anterior hypothalamus, whereas most HA-immunoreactive cell
bodies aggregated in both the ventral and dorsal divisions of the
tuberomammillary nucleus (TMn), in the adjacent lateral hypothalamic
area, and in the perimammillary and supramammillary areas (Fig.
11). The ventral division of the
histaminergic TMn was more compactly organized than the dorsal
division. The HA-immunoreactive neurons, estimated to number 2500-3500
in the whole brain, were medium to large in size (15 × 30 µm),
mostly ovoid or polygonal in shape, and possessed two to four
prominent, long, thick processes. The majority of the HA-immunoreactive
perikarya showed strong immunoreactivity, although a few were
moderately stained (Fig. 11).
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Figure 11.
Distribution of histamine-immunoreactive (HA-IR)
neurons in the mouse hypothalamus. Photomicrographs of frontal sections
showing HA-IR neurons in the mouse hypothalamus visualized using the
immunofluorescent CyTM3 method. Note the presence of HA-IR
cell bodies and fibers in the HDC+/+ mouse brain (left)
and their absence in the HDC / mouse (right).
3V, Third ventricle; CM, mammillary
corpus; f, fornix; ipf, interpeduncular
fossa; LH, lateral hypothalamic area;
Mre, mammillary recess; PH, posterior
hypothalamic area; SuM, supramammillary area;
TMv and TMd, tuberomammillary nucleus,
ventral and dorsal divisions. Scale bars, 100 µm.
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HA-immunoreactive fibers and terminal-like dots were detected in
virtually all brain regions. For example, numerous fine varicose fibers
were present in the various neocortical areas, hippocampal formation,
basal forebrain, thalamus, preoptic/anterior and posterior hypothalamus
including the perifornical area, and the forebrain and brainstem
aminergic and cholinergic structures, such as the substantia
innominata, ventral tegmental area of Tsai, mesopontine tegmentum,
raphe nuclei, and locus coeruleus (Figs. 11,
12). Omission of anti-HA antibody or
pre-incubation of the sections with excess exogenous free or
ovalbumin-conjugated HA resulted in no immunolabeling of any part of
the mouse brain (data not shown), demonstrating the specificity of
labeling. Examples of the presence of HA-immunoreactive fibers in some
of these structures are shown in Figure 12.
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Figure 12.
Distribution of histamine-immunoreactive (HA-IR)
cell bodies and fibers in the mouse CNS. Photomicrographs of frontal
sections showing HA immunoreactivity visualized using immunofluorescent
CyTM3 (dark-field photomicrographs) or PAP (light-field
photomicrographs of sections counterstained with neutral red). Note, in
HDC+/+ mice, but not HDC/ mice, the presence of HA-IR fibers in the
primary somatosensory cortex (S1), the diagonal band of
Broca (BDB), and the substantia innominata
(SI) of the basal forebrain, the geniculate
nucleus of the thalamus (MG), and brainstem structures,
such as the dorsal raphe nucleus (DR), substantia nigra
(SN), locus coeruleus (LC), and
laterodorsal tegmental nucleus (LDT). Also note
the presence of HA-IR neurons in the brain section through the
tuberomammillary nucleus (TMv) in the HDC+/+ mouse but
not the HDC/ mouse. Aq, Aqueduct of Sylvius;
mlf, medial longitudinal fasciculus; cp,
cerebral peduncle; scp, superior cerebellar peduncle.
Scale bars, 100 µm.
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HDC/ mice
In contrast to the dense HA immunoreactivity present in the HDC+/+
brains, no HA immunostaining was found throughout the HDC/ brains
using either immunofluorescent or PAP techniques, and no HA-immunoreactive perikarya, dendrites, fibers, or terminal-like dots
were identified in the TMn and the adjacent posterior hypothalamus or
elsewhere in the brain (Figs. 11, 12).
Because the lack of the neurotransmitter, HA, TMn neurons in HDC/
mice can no longer be qualified as histaminergic neurons. However, as
demonstrated by neutral red counterstaining (Fig. 12) or
immunohistochemistry of type B monoamine oxidase (data not shown), a
marker for TMn neurons (Lin et al., 1993), both dorsal and ventral
divisions of the TMn, although nonimmunoreactive with anti-HA antibody,
seemed to be intact in these mice, without obvious visual difference in
either the number of neurons or their morphology (see example from the
ventral division in Fig. 12). Finally, no visually apparent structural
changes were seen in the brain sections examined. Because the number
and morphology of TMn neurons under the light microscopy appeared to be
unchanged in HDC/ mice, the nature of the functional change after
HDC disruption and the neurotransmitter(s), if any, which replaces HA
remains to be determined.
Summary of the principal findings
The present study reveals the absence of detectable brain
HA-immunoreactive neurons and the absence of response to administration of HA-related agents in the PCR-confirmed HDC/ mice. Moreover, we
have shown that the sleep-wake cycle of these mice was affected both
quantitatively and qualitatively. On the one hand, these mice exhibited
an increase in the daily amount of PS (+23%) and a deficit in W just
before and after lights-off. On the other hand, their cortical-EEG
showed a reduced SWS/W power ratio and a significant increase in frequencies (0.8-2.5Hz) and a deficit of -rhythms (3-9Hz) during
W. These changes are likely to have an effect on the animal's
behavior, because the HDC/ mice presented clear signs of sedation,
manifested as a significant decrease in sleep latencies after several
behavioral stimuli, and more importantly, unlike normal mice, in being
unable to remain awake in a new environment.
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DISCUSSION |
Several lines of evidence from our study indicated that the
cortical EEG and behavioral signs seen in HDC/ mice are caused by
the lack of HA synthesis. First, the mouse strain, sex, and age and the
experimental conditions were identical for the wild-type and KO mice,
and the only correlation with the observed effects was with the mouse
genotype. Because our study used inbred mice, the genomic background of
the two genotypes was identical, except for the HDC gene. The small
interindividual SDs for the sleep-wake stages within genotype
also indicated that each group was homogenous (Fig. 1). Second, in
HDC+/+ mice, -FMH (HDC inhibitor) produced the same changes seen in
untreated HDC/ mice, whereas ciproxifan (H3 receptor antagonist
that enhances HA transmission; Ligneau et al.,1998) elicited cortical
activation. Both drugs had no effect in HDC/ mice, indicating their
dependence on the availability of HDC and endogenous HA, which are
lacking in HDC/mice. Although, because of absorption from the
digestive tract, trace amounts of HA may be present in the HDC/
brain (Ohtsu et al., 2001), this is likely to be non-neuronal and
outside the blood-brain barrier, because HA does not pass through the
barrier (Schwartz et al., 1991) and because histaminergic cells were
not detectable in the TMn and throughout the HDC/ brain, indicating
the absence of neuronal-HA. Finally, peripheral injection of HA had no
effect on either the EEG or sleep-wake cycle in either genotype,
indicating that cortical arousal did not depend on peripheral HA. These
results, together with the established role of histaminergic neurons in arousal, leave little doubt that the loss of HA from TMn cells is
responsible for the effects on the cortical EEG and behavioral states
in HDC/ mice.
Histaminergic neurons and spontaneous waking
Like other KO-mice that are viable and develop normally, e.g.,
those lacking orexin (Chemelli et al., 1999), 5HT1B receptors (Boutrel et al., 1999), or HA-H1-receptors (Lin et al., 2001, 2002),
the HDC/ mice exhibited a circadian rhythm characteristic of
rodents. Moreover, there is no major change in the daily amount of
spontaneous W(<-15%/24 hr) in any of the above KO mice, even though
each of the abolished systems is thought to regulate W. These results
suggest that, in these mice, the molecular mechanisms and neuronal
substrates essential to sleep-wake alternation are not fundamentally
impaired by the disruption, during early-embryogenesis, of a gene
regulating sleep-waking and that brain plasticity functions in such a
way that an adaptive mechanism is already elaborated during
early-ontogenesis of the sleep-wake cycle to compensate for the
functional loss. Although chronic abolition is quite different to acute
destruction in terms of brain plasticity, these results are comparable
to those obtained by lesioning. Indeed, destruction of one of the brain
structures involved in cortical activation causes a transient reduction
in W and quite prompt restoration of approximately normal sleep-wake
cycle (Villablanca, 1974; Buzsaki et al., 1988; Vanderwolf and Stewart,
1988; Webster and Jones, 1988; Denoyer et al., 1991). W is a functional
state allowing the performance of vital behavior and high brain
functions. The maintenance of the cerebral cortex in this highly
complex state necessitates the convergent and divergent activity of a
large ascending network extending from the medulla to the forebrain and
involving several neurotransmitters (Moruzzi, 1972; Steriade, 1991;
McCormick, 1992; Jones, 2000; Lin, 2000). Although it is generally
presumed that a chronic loss of one system could be compensated by
increased activity of the others to maintain a behavioral state as
important as W, the mechanisms involved remain unknown.
Despite the lack of major quantitative change in the daily spontaneous
W, qualitative aspects of W in HDC/ mice were markedly affected.
First, during W, these mice exhibited increased power in rhythms,
the most important sign of cortical inactivation occurring
predominantly during natural SWS. Secon |
TOP
ABSTRACT
INTRODUCTION
