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The Journal of Neuroscience, April 15, 1999, 19(8):3057-3072
Differential c-Fos Expression in Cholinergic, Monoaminergic, and
GABAergic Cell Groups of the Pontomesencephalic Tegmentum after
Paradoxical Sleep Deprivation and Recovery
Karen J.
Maloney,
Lynda
Mainville, and
Barbara E.
Jones
Department of Neurology and Neurosurgery, McGill University,
Montreal Neurological Institute, Montreal, Quebec, H3A 2B4, Canada
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ABSTRACT |
Multiple lines of evidence indicate that neurons within the
pontomesencephalic tegmentum are critically involved in the generation of paradoxical sleep (PS). From single-unit recording studies, evidence
suggests that unidentified but "possibly" cholinergic tegmental
neurons discharge at higher rates during PS than during slow wave sleep
or even waking and would thus play an active role, whereas
"presumed" monoaminergic neurons cease firing during PS and would
thus play a permissive role in PS generation. In the present study
performed on rats, c-Fos immunostaining was used as a reflection of
neuronal activity and combined with immunostaining for choline
acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH),
or glutamic acid decarboxylase (GAD) for immunohistochemical identification of active neurons during PS recovery (~28% of
recording time) as compared with PS deprivation (0%) and PS control
(~15%) conditions. With PS recovery, there was a significant
increase in ChAT+/c-Fos+ cells, a significant decrease in Ser+/c-Fos+
and TH+/c-Fos+ cells, and a significant increase in GAD+/c-Fos+ cells. Across conditions, the percent PS was correlated positively with tegmental cholinergic c-Fos+ cells, negatively with raphe serotonergic and locus coeruleus noradrenergic c-Fos+ cells, and positively with
codistributed and neighboring GABAergic c-Fos+ cells. These results
support the hypothesis that cholinergic neurons are active, whereas
monoaminergic neurons are inactive during PS. They moreover indicate
that GABAergic neurons are active during PS and could thus be
responsible for inhibiting neighboring monoaminergic neurons that may
be essential in the generation of PS.
Key words:
paradoxical sleep; c-Fos expression; cholinergic; serotonergic; noradrenergic; GABAergic; sleep-wake states
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INTRODUCTION |
Since early studies using
transections and lesions, the pontomesencephalic tegmentum has been
known to be critical for the generation of paradoxical sleep [(PS), or
rapid eye movement (REM) sleep] (Jouvet, 1962 , 1972). Pharmacological
evidence had indicated that acetylcholine (ACh) was important for the
appearance of PS (Domino et al., 1968), and injections of the
cholinergic agonist carbachol into the pontomesencephalic tegmentum was
shown to elicit a state similar to natural PS (George et al., 1964).
ACh was also known to be important for wakefulness, and enhancing ACh
levels with acetylcholinesterase (AChE) inhibitors elicited a waking state (Domino et al., 1968) unless monoamines were first depleted with
reserpine, in which case, it elicited PS (Karczmar et al., 1970). These
early pharmacological studies thus suggested that cholinergic systems,
particularly within the pontomesencephalic tegmentum, were involved in
the generation of PS but were also involved in waking and could only
elicit PS when monoaminergic systems were inactivated.
After immunohistochemical localization of the cholinergic neurons in
the pontomesencephalic tegmentum (Mesulam et al., 1983; Jones and
Beaudet, 1987), it was demonstrated that neurotoxic lesions of these
neurons resulted in the loss of PS (Jones and Webster, 1988; Webster
and Jones, 1988; Jones, 1991b). Single-unit recording within the
cholinergic cell area, including the laterodorsal and pedunculopontine
tegmental nuclei, found cells that discharged at higher rates during PS
than during slow wave sleep (SWS) and some that fired at even higher
rates during PS than during waking (El Mansari et al., 1989; Steriade
et al., 1990a; Kayama et al., 1992). In contrast, presumed serotonergic
raphe neurons and presumed noradrenergic locus coeruleus neurons were
found to fire at their lowest rates or cease firing altogether during
PS (Hobson et al., 1975; McCarley and Hobson, 1975; McGinty and Harper,
1976). These electrophysiological results supported the hypothesis that
PS is generated by an active involvement of cholinergic neurons, considered to be "PS-on" cells, and a permissive role of
monoaminergic neurons, considered to be "PS-off" cells (McCarley
and Hobson, 1975; Sakai, 1988; McCarley et al., 1995). The possibility
was also raised that the cessation of firing by the monoaminergic neurons could be caused by active inhibition by local GABAergic neurons, which are distributed within these cell groups and neighboring areas (Jones, 1991a,b,c, 1993; Ford et al., 1995). The validation of
these hypotheses depends, however, on the chemical identification of
the recorded units, which has not yet been possible in naturally sleeping-waking animals.
Another method of studying neuronal activity, which permits
immunohistochemical identification of the active cells, is by examination of c-Fos, the product of the immediate early gene that is
expressed in association with neuronal discharge and entry of
Na+ and Ca2+ ions (Morgan and
Curran, 1986; Dragunow and Faull, 1989). c-Fos expression combined with
immunohistochemical staining for cholinergic and monoaminergic neurons
has been used in cats in the study of neurons active during
carbachol-induced PS (Shiromani et al., 1992; Yamuy et al., 1995,
1998), yet this state may fundamentally differ from naturally generated
PS. In the present study performed in rats, we sought to examine c-Fos
expression in association with naturally enhanced PS during rebound
from PS deprivation (Mendelson, 1974). Dual immunostaining for c-Fos
protein and neurotransmitters or their enzymes was used for relative
assessment of activity in cholinergic, serotonergic, noradrenergic, and
GABAergic neurons in the pontomesencephalic tegmentum of animals under
conditions of PS recovery, deprivation, and control (Maloney and Jones,
1997).
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MATERIALS AND METHODS |
Animals and surgery. Sixteen male Wistar rats
(Charles River, Montreal, Quebec, Canada), weighing ~225 gm, were
operated under barbiturate anesthesia (Somnotol; 67 mg/kg, i.p.) for
the implantation of chronically indwelling electrodes. For the
electroencephalogram (EEG), stainless steel screws were threaded into
holes drilled in the skull so that the screws were in gentle contact
with the dura. They were placed over the left and right retrosplenial, anterior frontal, parietal, and occipital cortices, as described previously (Maloney et al., 1997). One electrode was placed in the
frontal bone rostral to the frontal lobes to be used as a reference for
monopolar recording from each cortical lead and one in the occipital
bone over the cerebellum to be used as a ground electrode. For the
electromyogram (EMG), two stainless steel loops were inserted into the
muscles of the neck. All leads were connected to a miniature (12 lead)
plug that was cemented to the skull. Animals were allowed 2 or 3 d
recovery from surgery in the animal room before being placed in
recording chambers for the duration of the experiment.
Recording and experimental procedures. For recording and
experimentation, each rat was placed in a Plexiglas box that was contained within a larger electrically shielded recording chamber. The
rat was connected to a cable that was attached to a commutator and
suspended with a balanced boom to allow free movement of the animal
within the box. During the baseline day and in the control condition,
the floor of the box was covered with woodchips. The animal had
ad libitum access to food and water in containers that hung within easy reach on the sides of the box. As was the case in the
animal room, a 12 hr light/dark cycle was maintained in the recording
room (with lights on from 7:00 A.M. to 7:00 P.M.). The rat was placed
in the recording box and connected to the cable 3 d before
baseline recording to allow for habituation to the recording environment.
The EEG and EMG signals were amplified using a Grass model 78D
polygraph and subsequently sent to a computer (ALR 386SX) for analog-to-digital conversion, filtering, and storage on hard disk with
the aid of Stellate Systems (Montreal, Quebec, Canada) computer software, as described previously (Maloney et al., 1997).
PS deprivation was performed using the flower pot technique that has
previously been shown to cause a fairly selective deprivation of PS in
rats (Mendelson, 1974). It was also shown to not be associated with
significant changes in adrenal gland weights (Mendelson, 1974), thus
not producing a severe level of stress in the animals. Each rat was
placed on an inverted flower pot that was just large enough (~6.5 cm
in diameter) to hold the animal. The flower pot was surrounded by water
that filled the Plexiglas box to within 1 cm of the surface of the
inverted pot. In this situation, the animal could engage in SWS
but not PS, because the loss of muscle tonus that occurs with PS onset
causes the animal to fall into the water and awaken. Food and water
containers were positioned to be easily accessible to the animal on the
flower pot. Under these experimental conditions, it was determined in
preliminary recording experiments (involving four rats operated for
implantation of electrodes and tested in the recording and experimental
paradigm) that after the first 24 hr on the flower pots during which a
certain degree of habituation to the experimental situation occurred, SWS appeared in ostensibly normal amounts but PS remained suppressed, producing a relatively selective deprivation of PS in the second 24 hr
deprivation period and selective rebound of PS after the deprivation.
Accordingly, an ~48 hr deprivation period on the flower pots was
selected for the experimental paradigm.
The experimental protocol was performed over a 4 d period in three
groups of four rats (Fig. 1). Recordings
were performed in the afternoon (~12:00-3:00 P.M.) for the four
consecutive days. On the first day, a baseline recording was performed
on all animals. On the remaining 3 d of the experiment, the
"condition" was varied for the three different groups: PS control
(PSC), PS deprivation (PSD), and PS recovery (PSR). (1) For the control
condition, the PSC animals remained on a bed of woodchips in their
recording boxes for the 4 d. During these days, they were left
undisturbed, except in the morning (~10:00-10:30 or ~11:00-11:30
A.M.) when their boxes were cleaned, and food and water were
replenished. At the termination of the experiment on day 4, the PSC
animals were anesthetized for perfusion (at ~3:00 or 3:30 P.M.) after the afternoon recording period (Fig. 1). (2) For the deprivation condition, the PSD animals were placed on flower pots for the second,
third, and fourth days of the experiment. On these days, they were
removed from their flower pots in the morning (~10:00-10:30 or
~11:00-11:30 A.M.) while their boxes were being cleaned, and they
were allowed to run around the larger dry recording chamber. On day 4 after the recording period, the PSD animals were anesthetized for
perfusion (at ~3:30 P.M.), having been in the deprivation condition
for ~53 hr (Fig. 1). (3) For the recovery condition, the PSR animals
were also placed on flower pots for the second, third, and fourth days
like the PSD animals. Similarly, they were removed from their cages
each morning (~10:00-10:30 A.M.) while their boxes were cleaned.
However, on day 4 after cage cleaning and after ~50 hr of PS
deprivation, the animals were returned to a dry bed of woodchips in
their recording boxes to allow for recovery of PS. To maximize the
recovery during the final recording period 3 hr before perfusion, the
cage cleaning was performed at ~11:00 A.M. on day 4, and the animals
were returned to the dry box at ~11:30 A.M., thus allowing ~30 min
exploration and grooming before sleep onset and recording. All three
groups of animals commonly tended to remain awake and active during the ~30 min period after cage cleaning and handling (from
11:00-11:30 on day 4). After the recording period, the animals were
anesthetized for perfusion (at ~3:00 P.M.), having been in the PS
recovery condition for ~3 hr after PS deprivation of ~50 hr (Fig.
1).
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Figure 1.
Diagram illustrating time course of
treatments for the three different experimental conditions: PSC, PSD,
and PSR. Whereas the PSC group remained on a bed of woodchips,
the PSD and PSR groups were placed on inverted flower pots surrounded
by water for ~53 or ~50 hr deprivation of PS (Days
2-4) in the recording chambers. The PSR
group was returned to the woodchips to allow ~3 hr recovery of PS.
Animals were anesthetized and perfused ( ) at the end of the control,
deprivation, or recovery periods (on day 4). EEG recording was
performed each day for 3 hr in the afternoon. Animals were maintained
on a 12 hr light/dark cycle (with lights on from 7:00 A.M. to 7:00
P.M., as indicated by light shading). See Materials and Methods for
further details.
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The experiments were conducted using two recording chambers and thus on
two animals at one time, running pairs of PSD-PSR or PSC-PSC animals.
Because the recovery condition was considered the most constrained with
regard to time, the PSR animals were always anesthetized and perfused
first at ~3:00 P.M., whereas the paired PSD animals were anesthetized
and perfused second at ~3:30 P.M. For the PSC-PSC pairs, one PSC
animal was anesthetized at ~3:00 and the second at ~3:30 P.M. The
entire course of experiments involving six pairs of rats was conducted
over a 3 month period during the winter season.
Pilot animals and procedures. Before the recording and
experimental study described above, a pilot experiment was performed to
test the effectiveness of the experimental paradigm and
immunohistochemical revelation of c-Fos together with the
neurotransmitters or enzymes. A series of twelve Wistar rats (Charles
River), weighing ~225 gm, were submitted to the three different
experimental conditions described above, except that the pilot animals
were not operated for implantation of electrodes and not attached to
cables for recording. Another group of four rats were kept in the
animal room, two to a cage in the manner that they are typically
housed. These four groups of animals were anesthetized and perfused in pairs at ~3:00 or ~3:30 P.M. (as above).
Perfusion and fixation. The animals were killed under
barbiturate anesthesia (Somnotol; ~100 mg/kg) by intraaortic
perfusion of a fixative solution. The time between the barbiturate
injection and initiation of the perfusion was ~10 min. One liter of
3% paraformaldehyde and 0.2% picric acid in 0.1 M
phosphate buffer was perfused for fixation and followed by 250 ml of
10% sucrose in buffer. The brains were immersed in 30% sucrose
overnight to complete cryoprotection. The brains were frozen at
 50°C and stored at 80°C.
Immunohistochemistry. Coronal sections were cut at 25 µm
thickness on a freezing microtome. Up to six series of adjacent
sections were collected every 200 µm for immunohistochemical
processing. All immunohistochemistry was performed using the
peroxidase-antiperoxidase (PAP) technique (Sternberger, 1979),
according to previously published procedures (Gritti et al., 1993,
1997; Ford et al., 1995). For the immunostaining of c-Fos protein, an
anti-c-Fos antiserum from sheep (Cambridge Research Biochemicals,
Cheshire, UK) was used at a dilution of 1:3000. For immunostaining of
neurotransmitters or their enzymes, the following antibodies were used:
rabbit anti-choline-acetyl transferase (ChAT) antiserum (1:3000;
Chemicon International, Temecula, CA), rabbit anti-serotonin (Ser)
antiserum (1:30,000; Incstar, Stillwater, MN), rabbit anti-tyrosine
hydroxylase (TH) antiserum (1:15,000; Eugene Tech International,
Allendale, NJ), and rabbit anti-glutamic acid decarboxylase (GAD)
antiserum (1:3000; Chemicon International). Incubations with primary
antibodies were performed at room temperature overnight using a
Tris-saline solution (0.1 M) containing 1% normal donkey
serum (NDS) and after incubation with Tris saline containing 6% NDS
for blocking. For antibodies to ChAT and TH, Triton X-100 (0.2%) was
added to the incubation solutions. Appropriate secondary antisera and
PAP antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA)
were used after the respective primary antisera. In all brains, one
series of sections was immunostained for c-Fos alone using the brown,
floccular reaction product, 3,3' diaminobenzidine (DAB) as chromogen.
In other adjacent series processed from the pilot and experimental
brains, c-Fos was immunostained in combination with a neurotransmitter
or enzyme using a sequential procedure staining c-Fos in either the
first or second position. When in the first position, c-Fos was
revealed with DAB, and the neurotransmitter or enzyme was revealed with the blue granular reaction product, benzidine dihydrochloride (BDHC).
In one set (PSD-PSR-PSC), c-Fos was revealed with DAB intensified
with nickel (DAB-Ni2+) in the first position, and
the neurotransmitter or enzyme revealed with DAB in the second
position. In the major experimental series, the neurotransmitter or
enzyme was immunostained and revealed in the first position with DAB,
and c-Fos was immunostained and revealed in the second position with
BDHC. Controls in the absence of primary antibodies and in the presence
of normal sera were routinely run with every single and dual
immunostaining procedure to ensure the absence of nonspecific single or
dual immunostaining in the material. Brains from sets of PSD-PSR,
which were run together experimentally, were processed in the same
manner for immunohistochemistry together with an accompanying PSC brain.
In assessing the effectiveness of the experimental paradigm and
immunohistochemistry, qualitative examination of the material was
performed by one of the experimenters (B.E.J.) who had knowledge of the
experimental groups. In comparing the numbers of c-Fos immunostained
cells across the different conditions used in the pilot and
experimental studies, it was apparent that few to no cells were stained
in brains from animals housed in pairs in the animal colony, many cells
were stained in animals kept for 4 d under control conditions
housed individually in the recording chambers, and many more cells were
stained in animals that had been operated for implantation of
electrodes and were attached to recording cables for 4 d under
control conditions in the recording chambers. It was thus clear that
the base level of c-Fos expression in the pilot and more so
experimental animals was relatively high and could be attributed to the
degree of stress associated with isolation, a new environment, surgery,
and being tethered, despite allowing habituation to the recording
environment and recovery from surgery. c-Fos expression has been known
to be elevated in widespread areas in response to stress (Pezzone et
al., 1993; Chen and Herbert, 1995; Cullinan et al., 1995). The level of
c-Fos expression caused by these factors would however be the same for the three pilot and the three experimental groups, respectively, that
were being compared among themselves. Qualitative differences in c-Fos
immunostaining could be detected across the three different conditions
of the pilot and experimental groups, respectively, and appeared
systematic across these conditions in the two groups.
In the pilot series, c-Fos was immunostained in the first position, and
the neurotransmitter or enzyme was immunostained in the second
position. In this series, it was clear that more TH+ cells in the locus
coeruleus were c-Fos+ in the PSD group than in the PSR group. However,
it was difficult to clearly discern the ChAT and GAD immunostaining in
the second position and thus to appreciate changes in the number of
ChAT+ and GAD+ cells expressing c-Fos across the different groups.
Thus, for the major experimental series, the immunostaining procedure
was changed so that the neurotransmitter or enzyme was stained in the
first position (with DAB), and c-Fos was stained in the second position
(with BDHC). This procedure reduced the sensitivity of the c-Fos
immunostaining particularly in the TH+ cells and also in Ser+ cells, in
which the TH and serotonin staining was intense. However, it greatly
enhanced the immunostaining, and thus identification of cells
containing ChAT and GAD, in which ChAT and GAD staining was only
moderate and c-Fos staining thus less affected. Because brains were
processed in pairs from PSD-PSR animals and subsequently in a like
manner from a matching PSC animal, it could be determined that the
relative differences across groups were the same independent of the
immunostaining procedure, although the absolute numbers of
c-Fos-immunostained cells were lower with the c-Fos staining in the
second position.
Analysis of sleep-wake state data. The EEG was examined by
off-line analysis on computer screen and scored for sleep-wake state
by visual assessment of EEG and EMG activity in 20 sec epochs using
Eclipse software (Stellate Systems) for each 3 hr recording session
(~540 epochs). Epochs were scored as one of the three major states
(Wake, SWS, or PS) or transitional (t) stages between states: (1) wake marked by the presence of low-voltage fast activity associated with EMG tonus, (2) transition from wake into slow wave
sleep (tSWS) characterized by moderate amplitude slow or mixed slow, spindle and fast activity, (3) slow wave sleep (SWS) marked
by continuous high-amplitude slow activity, (4) transition from slow
wave sleep into PS (tPS) marked by a decrease in
high-amplitude slow activity and the appearance of spindles and theta
waves, or (5) PS characterized by a prominence of theta waves, in
addition to low-voltage fast activity, with low EMG activity.
The number of epochs scored in each state was calculated as a percent
of total epochs in the 3 hr recording session for each day. An overall
statistic was performed using a repeated measures ANOVA with two trial
factors ("state" and "day") and one grouping factor
("condition"). Data were further analyzed per state by repeated measures ANOVA tests with one trial factor (day) and one
grouping factor (condition). When a main effect of condition was
significant, post hoc tests were performed per day
across groups (PSR or PSD vs PSC; PSR vs PSD) using Fisher's pairwise comparison. In the case in which there was a significant difference between groups (condition), another test was performed to determine whether there was also a significant difference between days 2, 3, or 4 and day 1 in that group. These tests were performed per state and
condition, using a repeated measures ANOVA with one trial factor (day)
and post hoc tests performed between the experimental days (day 2, 3, or 4) and baseline day (day 1). The number of PS
episodes and the mean duration of these episodes were also calculated
for the baseline day 1 and recovery day 4 of each animal in the PSR
condition and analyzed by t test paired comparisons.
Fast Fourier Transform was performed using Rhythm software
(Stellate Systems) to determine power in EEG frequency bands for the 20 sec state-scored epoch data, as described previously (Maloney et al.,
1997). Frequency bands were set at the following ranges: delta,
1.5-4.0 Hz; theta, 4.5-8.5 Hz; and gamma, 30.5-58.0 Hz. The ratio of
theta/delta, which reflects theta rhythmicity on the EEG, was also
calculated and displayed. EMG amplitude was computed for the total
spectrum up to 58.0 Hz. Changes in frequency band activities across
experimental conditions were examined by statistical analysis of
activities from the right retrosplenial lead. Frequency band activity
was normalized for each rat according to the average amplitude values
per state in the baseline day. Statistical differences in frequency
band activity per state and condition were tested by repeated measures
ANOVA with one trial factor (day).
Analysis of immunohistochemical data. Sections were viewed
with a Leitz Orthoplan microscope equipped with an x/y
movement-sensitive stage and CCD camera attached to a computer. Single-
and dual-immunostained cells were mapped using a computer-based image
analysis system (Biocom, Paris, France) with a resident atlas of
sections through the pontomesencephalic tegmentum (Jones, 1995). The
experimenter (K.J.M.) mapping the cells did not have knowledge of the
experimental group (PSD, PSR, PSC) to which the individual brains
belonged. She was only given this information after all the data were
tabulated on computer spreadsheets, and the group condition was
inserted for the statistical analysis of the completed data set. Cell
counts were tabulated automatically within each nucleus or region,
including those of the cholinergic, serotonergic, and noradrenergic
cell groups and the adjacent central gray areas of the
pontomesencephalic tegmentum. Single c-Fos-immunostained cells were
mapped and counted unilaterally (in one or two sections) at one or two
representative of three stereotaxic levels corresponding approximately
to anterior (A) 0.5, A 0.1, and posterior (P) 0.3, depending on the
specific nucleus (Paxinos and Watson, 1986; Jones, 1995). To allow a
more thorough sampling of dual immunostained cells, which were less numerous than the single c-Fos-immunostained cells and represented the
focus of the present study, double-labeled cells were counted bilaterally at 200 µm intervals through the full rostrocaudal extent
of each cholinergic and serotonergic cell group and individual nucleus
(in four to six sections between ~A 1.1 and ~P 0.5 depending on the
specific nucleus) and through the rostral to midportion of the
noradrenergic cell group (for two or three sections, between ~P 0.1 and ~P 0.5 depending on the specific nucleus). GAD+/c-Fos+ cells were
counted on adjacent sections to ChAT+/c-Fos+, Ser+/c-Fos+, or
TH+/c-Fos+ cells in the same cell groups and nuclei, except the locus
coeruleus (where too few GAD+ cells are located, (Ford et al., 1995))
and additionally in the rostral and caudal central gray areas
neighboring the dorsal raphe and locus coeruleus, respectively. The
bilateral cell counts for dual-immunostained cells were averaged per
section across the two sides. ANOVAs or ANCOVAs were performed on the cell counts across conditions in multiple sections per nucleus
per cell group (cholinergic, serotonergic, noradrenergic, or central
gray) per animal. Overall statistical differences in the number of
cells caused by condition were examined in each cell group by one-way
ANCOVA with condition as the grouping factor and nucleus, section, and
animal as covariates. Statistical differences in the number of cells in
individual nuclei within each cell group were subsequently examined
using a one-way ANCOVA with condition as grouping factor and section
and animal as covariates. When there was a significant main effect,
differences in cell counts between individual conditions were analyzed
by post hoc analyses using Fisher's pairwise
comparisons (with significance level set at p  0.05).
For tabular presentation and regression analysis, the total number of
labeled cells was calculated for each nucleus and cell group by adding
(absolute or averaged) unilateral counts across sections in individual
animals. Correlations between total number of labeled cells counted per
nucleus or cell group and the percent PS, SWS, or wake were performed
by multiple linear regression analyses with animal as a covariate. All
statistics were performed using Systat for Windows (Evanston,
Illinois). Figures were prepared for publication using CorelDraw
(Ottawa, Ontario).
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RESULTS |
Sleep-wake states
The PS deprivation procedure, which commenced on day 2 of the
experimental paradigm for animals of the deprivation and recovery conditions (Fig. 1), was effective in producing a near complete elimination of PS, as well as a significant decrease in the
transitional state into PS (tPS), as measured during 3 hr
recording periods on days 2 and 3 in the deprivation and recovery
conditions (Table 1, PSD, PSR). The
decrease in PS and tPS was associated with a significant
increase in waking. Although SWS was reduced, particularly on the first
deprivation day, it was not significantly less than that in the PSC
group during the deprivation days (Table 1).
In comparing the sleep-wake states across the different conditions on
day 4 when the recovery group was removed from the deprivation condition (Fig. 1), it was apparent that there was a relatively selective deprivation of PS in the deprivation condition (PSD) and a
relatively selective recovery of PS in the recovery condition (PSR), as
compared with the control condition (PSC) in the final 3 hr before
anesthesia and perfusion (Table 1). Thus, after ~53 and 50 hr of
deprivation, respectively, PS represented 0% in the deprivation
condition and ~28% in the recovery condition, as compared with
~15% in the control condition. SWS was less in the deprivation condition than in control, but not significantly so; wake was significantly greater in the deprivation condition (Table 1). SWS was
significantly greater in the recovery condition than in control or
deprivation, however it was not significantly different from baseline
within the same group (Table 1, as indicated by parentheses). As
compared with both control and baseline conditions, therefore, PS was
the one state that was commonly altered in the two experimental
conditions, being significantly decreased in the deprivation and
significantly increased in the recovery condition.
Although the experimental procedure was effective in producing a
relatively selective deprivation and recovery of PS, it did so without
causing major changes in the EEG characteristics of the extant states
of wake and SWS during deprivation, as well as of PS during recovery
(Fig. 2). According to visual inspection of the record and quantitative assessment of frequency band activity, the EEG was relatively unchanged during waking and was characterized, as in baseline and control conditions, by high-frequency gamma activity
and theta waves recorded from limbic cortex [Figs. 2, right
retrosplenial (RRS), 3, 4]. Across deprivation days, though, there was a progressive increase in gamma activity during waking that
was significant for the deprivation condition (F = 6.068; df = 3; p < 0.05). The EEG during SWS was
relatively unaltered and characterized as in baseline and control
conditions by high-amplitude delta waves during deprivation and
recovery (Figs. 2-4). There were no significant differences in
amplitude of delta activity in SWS. PS during recovery (day 4) appeared
similar in its EEG characteristics to that during baseline (Figs. 2, 4,
Day 1). Gamma and theta activities were not
quantitatively different from those in baseline PS. The change in PS
during recovery was thus measured as being only in amount (Fig. 4),
which particularly reflected consistently increased duration of PS
episodes (2.44 ± 0.49 vs 1.84 ± 0.35 min, mean ± SEM;
t = 12.5; df = 3; p < 0.05 with paired comparison of recovery to baseline values), in addition to
frequently increased numbers of PS episodes (23 ± 4.3 vs 17 ± 2.7; t = 1.58; df = 3, not significant).
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Figure 2.
EEG and EMG associated with wake, SWS, and PS on
day 1 (baseline) and day 4 from two representative animals within the
PSD (left) and PSR (right) groups. There
is no apparent difference in EEG activity during wake or SWS in either
group or during PS in the recovery group between days 1 and 4. The EEG
was recorded from the RRS cortex.
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Figure 3.
Hypnogram showing sleep-wake states (top
row, with PS also indicated by black bars
underneath; scored per 20 sec epoch during the 3 hr recording period)
over the four experimental days in one representative animal of the PSD
condition. Activity in the gamma (30.5-58.0 Hz) and delta (1.5-4.0
Hz) frequency bands and the ratio of theta (4.5-8.5 Hz) to delta
(Th/De, indicative of theta activity) are
shown for EEG. Total activity (1.5-58 Hz) is shown for EMG. Parallel
increases in gamma and theta reflect cortical activation during wake
and PS, which is also accompanied by low EMG activity, whereas high
delta activity reflects SWS. Note on days 2, 3, and 4, the persistence
of SWS marked by high-amplitude delta, despite the deprivation of PS.
Activity displayed as amplitude units scaled to maximum.
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Figure 4.
Hypnogram showing sleep-wake states (top
row, with PS also indicated by black bars
underneath; scored per 20 sec epoch during the 3 hr recording period)
over the four experimental days in one representative animal of the PSR
condition. Activity in the gamma (30.5-58.0 Hz) and delta (1.5-4.0
Hz) frequency bands and the ratio of theta (4.5-8.5 Hz) over delta
(Th/De, indicative of theta activity) are
shown for EEG. Total activity (1.5-58 Hz) is shown for EMG. Parallel
increases in gamma and theta reflect cortical activation during wake
and PS, which is also accompanied by low EMG activity, whereas high
delta activity reflects SWS. Note on days 2 and 3, the persistence of
SWS marked by high-amplitude delta, despite the deprivation of PS. The
recovery and rebound of PS is evident on day 4 by the presence of
high-amplitude gamma and theta activity with diminished EMG. Activity
displayed as amplitude units scaled to maximum.
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c-Fos+ expression in cell groups of the
pontomesencephalic tegmentum
The number of single-labeled c-Fos+ cells varied significantly as
a function of the experimental condition in most cell groups (Table
2, c-Fos+). However, whether there was an
increase or decrease in the PS recovery condition depended on the
individual cell group. In the cholinergic cell group, including its
four nuclear subdivisions, c-Fos+ cells were greater in the PSR
condition than in the PSD or PSC conditions (Table 2, Sum). In the
serotonergic cell group, including its two nuclear subdivisions, c-Fos+
cells were not consistently different in the recovery condition. In the
noradrenergic cell group, including its two nuclear subdivisions, c-Fos+ cells were lower in the recovery condition than in the deprivation condition. In the central gray areas, which lie adjacent to
the cholinergic and monoaminergic cell groups, c-Fos+ cells were
greater in the PS recovery condition than in the deprivation and
control conditions. The different variations according to condition in c-Fos+ cells across the different cell groups appeared to
depend on the predominant cell types located in those regions, as could
only be fully appreciated by dual immunostaining for c-Fos and specific
neurotransmitter or synthetic enzyme (Figs. 5-8).
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Table 2.
Number of identified single-labelled, c-Fos+ neurons, or
double-labelled, ChAT+/c-Fos+, Ser+/c-Fos+, TH+/c-Fos+, and GAD+/c-Fos+
neurons in the cholinergic, serotonergic, noradrenergic and adjacent
central gray cell groups of the pontomesencephalic tegmentum in PSC,
PSD, and PSR groups
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Figure 5.
Photomicrographs of sections dual-immunostained
for c-Fos (blue granular chromogen, BDHC) and ChAT
(A), GAD (B), serotonin
(C), or TH (D)
(brown chromogen, DAB). Black arrowheads
indicate double-labeled cells, and white arrowheads
indicate examples of adjacent single-labeled c-Fos+ cells. Scale, 25 µm.
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Figure 6.
Computerized atlas figure through the
pontomesencephalic tegmentum (~A 0.5) showing the cholinergic cell
group (top) where ChAT+/c-Fos+ cells
(circles) and GAD+/c-Fos+ cells
(triangles) were mapped (bottom) in
representative animals from PSD (left) and PSR
(right) groups. Note apparent increase in ChAT+/c-Fos+
cells and GAD+/c-Fos+ cells in the PSR condition compared with the PSD
condition. LDTg, Laterodorsal tegmental nucleus;
LDTgV, laterodorsal tegmental nucleus, ventral part;
PPTgM, pedunculopontine tegmental nucleus, medial part;
PPTgL, pedunculopontine tegmental nucleus.
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Figure 7.
Computerized atlas figure through the
pontomesencephalic tegmentum (~A 0.5) showing serotonergic cell group
and surrounding rostral central gray area (top) where
Ser+/c-Fos+ cells (asterisks) and GAD+/c-Fos+ cells
(triangles) were mapped (bottom) in
representative animals from the PSD (left) and PSR
(right) groups. Note the apparent decrease in
Ser+/c-Fos+ cells and increase in GAD+/c-Fos+ cells in the PSR
condition compared with the PSD condition. rCG, Rostral
central gray; DR, dorsal raphe nucleus;
MR, median raphe nucleus.
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Figure 8.
Computerized atlas figure through the
pontomesencephalic tegmentum (~P 0.3) showing noradrenergic cell
group and surrounding caudal central gray (top) where
TH+/c-Fos+ cells (squares) and GAD+/c-Fos+ cells
(triangles) were mapped (bottom) in
representative animals from PSD (left) and PSR
(right) groups. Note the apparent decrease in TH+/c-Fos+
cells and increase in GAD+/c-Fos+ cells in the PSR condition compared
with the PSD condition. cCG, Caudal central gray;
LC, locus coeruleus; SubCA, subcoeruleus
nucleus,  part.
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In the cholinergic cell group, ChAT-immunostained cells expressing
c-Fos (Fig. 5A) varied significantly as a function of
condition (Table 2, ChAT+/c-Fos+). The sum of ChAT+/c-Fos+ cells was
greater in the recovery condition than in the deprivation and control conditions (Table 2, Sum). The greater number of ChAT+/c-Fos+ cells in
the recovery condition as compared with the deprivation condition was
apparent across the cholinergic nuclei of the pontomesencephalic tegmentum (Fig. 6, LDTg, LDTgV,
PPTgM, PPTgL). Within individual nuclei, these
differences were statistically significant for the laterodorsal
tegmental nucleus (LDTg), in which the number of ChAT+/c-Fos+ cells in
the recovery condition was higher than in the deprivation condition,
and for the pedunculopontine tegmental nucleus, medial part (PPTgM), in
which the number of ChAT+/c-Fos+ cells in the recovery condition was
higher than in both the deprivation and control conditions (Table 2).
Within the LDTg, the number of cells in the deprivation condition was
also significantly lower than in the control condition. Across
conditions, the number of ChAT+/c-Fos+ cells within the LDTg was
significantly positively correlated with the percent time spent in PS
during the final 3 hr recording period (r = 0.68; Fig.
9). It was not significantly correlated
with the percent SWS but was significantly negatively correlated with
the percent waking (r = 0.73; df = 11;
p < .05). With stepwise backward removal of variables
in a multiple regression linear model, removal of the waking variable
did not eliminate the significant correlation of PS with the number of
ChAT+/c-Fos+ cells.
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Figure 9.
Numbers of ChAT+/c-Fos+ and GAD+/c-Fos+ neurons in
the LDTg, Ser+/c-Fos+, and GAD+/c-Fos+ neurons in the DR, and
TH+/c-Fos+ neurons and GAD+/c-Fos+ neurons in the LC and neighboring
cCG, respectively, for the three different groups, PSC, PSD, and PSR.
As according to the statistics detailed in Table 2, there was a
significant main effect of condition in every case shown. According to
post hoc tests, there were significant differences
between individual conditions as indicated: PSR or PSD versus PSC,
*p 0.05; PSR versus PSD, p 0.05. The inserts in the top left
corners show linear regression plots and coefficients of cell numbers
with the percent of PS across conditions. The scale of the
x-axis is consistent for all boxes and represents
percent PS (0-40%), as indicated. The scales of the
y-axes correspond to the number of cells, as indicated
in the respective full scale y-axis for each cell type
and nucleus. Correlations between percent time spent in PS and the
number of double-labeled cells in each nucleus were examined by
multiple linear regression with animal as a covariate
(*p 0.05, df = 11). See Table 2 and
Materials and Methods for other details.
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In the serotonergic cell group, Ser-immunostained cells expressing
c-Fos (Fig. 5C) varied significantly as a function of
condition (Table 2, Ser+/c-Fos+). The number of Ser+/c-Fos+ cells was
significantly lower in the recovery condition than in the deprivation
and control conditions (Table 2, Sum). The lower number of Ser+/c-Fos+
cells in the recovery condition as compared with the deprivation
condition was apparent in both the dorsal and median raphe nuclei (Fig. 7, DR, MR). Within both nuclei, the difference
between the recovery condition and deprivation and control conditions
was significant (Table 2). Across conditions, the number of Ser+/c-Fos+
cells within the DR was significantly negatively correlated with the percent time spent in PS during the final 3 hr recording period (Fig.
9; r = 0.73). It was not significantly correlated
with either the percent SWS or waking.
In the noradrenergic cell group, TH-immunostained cells expressing
c-Fos (Fig. 5D) varied significantly as a function of
condition (Table 2, TH+/c-Fos+). The number of TH+/c-Fos+ cells was
significantly lower in the recovery condition than in the deprivation
condition (Table 2, Sum). The lower number of TH+/c-Fos+ cells in the
recovery condition as compared with the deprivation condition was
apparent in both the locus coeruleus and subcoeruleus, part (Fig.
8, LC, SubCA). Within both nuclei, the difference
between the recovery condition and deprivation condition was
significant (Table 2). In addition, for both the sum and LC, TH+/c-Fos+
cells were significantly greater in the deprivation condition than in
the control condition (Table 2, Sum and LC). Across conditions, the
number of TH+/c-Fos+ cells within the LC was significantly negatively
correlated with the percent time spent in PS during the final 3 hr
recording period (Fig. 9; r = 0.61). It was not
significantly correlated with the percent SWS but was significantly
positively correlated with the percent waking (r = 0.60; df = 11; p < 0.05). With stepwise backward
removal of variables in a multiple regression linear model, removal of
the waking variable did not eliminate the significant correlation of PS
with the number of TH+/c-Fos+ cells.
In all cell groups, including the central gray areas adjacent to the
cholinergic and monoaminergic cell groups, GAD-immunostained cells
expressing c-Fos were evident (Fig. 5B) and varied
significantly as a function of condition (Table 2, GAD+/c-Fos+). The
number of GAD+/c-Fos+ cells was significantly higher in the recovery condition than in the deprivation and control conditions in all regions
(Table 2, Sum). The higher number of GAD+/c-Fos+ cells in the recovery
condition as compared with the deprivation condition was apparent in
all cell groups and nuclei of the pontomesencephalic tegmentum where
GAD+ cells are distributed (Figs. 6-8). In every one of these nuclei,
the number of GAD+/c-Fos+ cells was consistently higher in the recovery
condition than in the deprivation and control conditions (Table 2). In
none of the groups or nuclei were there significant differences between
the deprivation and control conditions (Table 2). Across conditions,
the number of GAD+/c-Fos+ cells was significantly positively
correlated with the percent time spent in PS in all nuclei, as
illustrated for the cells in the LDTg (r = 0.85), the
DR (r = 0.92), and the caudal central gray adjacent to
the LC (Fig. 9; r = 0.70). It was not significantly correlated with either the percent SWS or waking.
| |
DISCUSSION |
The present results demonstrate that during PS rebound, the number
of c-Fos-expressing cholinergic cells is increased, whereas the numbers
of c-Fos-expressing monoaminergic neurons are decreased, suggesting a
reciprocal change in the activity of these cell groups. Moreover, the
number of GABAergic cells expressing c-Fos during rebound is increased,
suggesting that they may also be active during PS and involved in
suppressing the activity of surrounding monoaminergic cells.
The changes in c-Fos expression are interpreted here as reflecting
changes in neuronal activity associated with the different experimental
conditions. It must be mentioned, nonetheless, that such changes may
also reflect changes in other cellular processes that can be stimulated
by chemical messengers independent of neuronal discharge, although also
dependent on changes in intracellular calcium (Morgan and Curran,
1986).
Cholinergic cell group
PS recovery resulted in increased numbers of cholinergic neurons
expressing c-Fos in the laterodorsal and pedunculopontine tegmental
nuclei, and across conditions the percent PS was significantly positively correlated with their numbers, supporting the hypothesis that cholinergic tegmental neurons are actively involved in PS generation. As confirmed here, previous studies examining
single-labeled c-Fos+ cells reported increases within the cholinergic
cell area in association with enhanced PS (Merchant-Nancy et al., 1992; Shiromani et al., 1992; Yamuy et al., 1993). Examining dual
immunostaining for c-Fos and ChAT, one study also found increases in
the number of cholinergic neurons expressing c-Fos during
carbachol-induced PS in cats, although, as is also the case in the
present study, only a small proportion of the cholinergic cells were
c-Fos+ (Shiromani et al., 1996). Another more recent study failed to
confirm the latter result in cats (Yamuy et al., 1998). However,
because carbachol acts directly on target neurons of the cholinergic
cells (Vanni-Mercier et al., 1989; Jones, 1990), its pharmacological
effect would not depend on increased activity by the cholinergic cells.
In vivo electrophysiological studies have identified
slow-spiking neurons as possibly cholinergic neurons and reported that
these cells are PS-on cells, all discharging at higher rates during PS
than during SWS and some higher during PS than during waking (Sakai and
Jouvet, 1980; El Mansari et al., 1989; Steriade et al., 1990a,b; Kayama
et al., 1992). Recently, juxtacellular labeling with biocytin combined
with staining for NADPH-diaphorase has provided histochemical evidence
that such slow-spiking neurons are cholinergic (Koyama et al., 1998).
Electrophysiologically characterized neurons have moreover been shown
to be inhibited by carbachol microinjections (as "Carb-I PS-on"
neurons) in vivo (Sakai and Koyama, 1996), similar to the
response to carbachol documented on identified cholinergic cells
in vitro (Leonard and Llinas, 1994). Further evidence that
cholinergic tegmental neurons are PS-on cells comes from biochemical
studies showing that in the thalamus and the brainstem pontine and
medullary reticular formation, to which the cholinergic tegmental
neurons project (Jones and Webster, 1988; Pare et al., 1988; Jones,
1990), ACh release is greater during PS than during SWS and in the
brainstem, also greater than during waking (Kodama et al., 1990, 1992;
Becker et al., 1994; Williams et al., 1994; Leonard and Lydic,
1995).
PS rebound was also associated with an increase in the number of
GABAergic neurons expressing c-Fos within the cholinergic cell area,
and across conditions, the percent PS was positively correlated with
their number. A significant proportion of neurons in the cholinergic
cell area that show increased c-Fos expression in association with
naturally occurring PS (Merchant-Nancy et al., 1992; Shiromani et al.,
1992) and also with carbachol-induced PS (Yamuy et al., 1998) would
thus, as the latter study also showed, be noncholinergic and as the
present study shows, most likely GABAergic. GABAergic neurons are
codistributed with the cholinergic neurons in the laterodorsal and
pedunculopontine tegmental nuclei (Ford et al., 1995) and could
correspond in part to other electrophysiologically identified PS-on
cells, which display brief spikes and discharge rapidly (Sakai and
Jouvet, 1980; El Mansari et al., 1989; Steriade et al., 1990a,b; Koyama
et al., 1998). In contrast to the putative cholinergic, these presumed
noncholinergic, PS-on cells are excited by carbachol microinjections
("Carb-E PS-on" neurons) in vivo (Sakai and Koyama,
1996). According to the electrophysiological study of these presumed
noncholinergic Carb-E PS-on neurons and to the chemoneuroanatomical
study of the GABAergic neurons in the cholinergic cell area, some PS-on
GABAergic neurons could be projection neurons and project in parallel
with the cholinergic neurons into the forebrain (Ford et al., 1995).
Other GABAergic cells may be locally projecting neurons and innervate
cell bodies or dendrites of neighboring monoaminergic neurons (Jones,
1991a,b), which they could thus inhibit during PS. In either case, they would appear to be active in parallel and perhaps also in series with
the cholinergic neurons during PS.
Monoaminergic cell groups
In the dorsal and median raphe nuclei, PS recovery resulted in a
decrease in the number of serotonergic cells expressing c-Fos, supporting the claim that presumed serotonergic neurons decrease or
cease firing during PS (McGinty and Harper, 1976; Trulson and Jacobs,
1979). Although a recent study with carbachol-induced PS in cats found
no significant difference as compared with saline-injected controls in
numbers of c-Fos-expressing serotonergic neurons, this lack of
difference was also interpreted as being caused by a lack of discharge
by the serotonergic neurons during PS (Yamuy et al., 1995). Support for
the cessation of serotonergic neuronal discharge during PS comes from
biochemical studies that have shown a marked decrease in serotonin
release during PS (Portas et al., 1998). In the present study, PS
recovery also resulted in an increase in the number of GABAergic cells
expressing c-Fos in the raphe and central gray, revealing potentially
active inhibitory interneurons that could be responsible for the
suppression of serotonergic activity. These GABAergic cells could
represent a proportion of dorsal raphe nonserotonergic c-Fos-expressing
cells that were increased in number with carbachol-induced PS (Yamuy et
al., 1995). The GABAergic c-Fos-expressing neurons may correspond to
neurons that were originally considered to be inhibitory interneurons, based on their distinct discharge properties and response to
stimulation, which was reciprocal to that of the dorsal raphe
serotonergic neurons (Aghajanian et al., 1978). Presumed
nonserotonergic neurons have been recorded across the sleep-waking
cycle, and some reported to increase their discharge during PS (Sheu et
al., 1974; Shima et al., 1986; Kocsis and Vertes, 1992). GABA release
measured in the dorsal raphe has been reported to be higher during PS
than during SWS or waking (Nitz and Siegel, 1997a). Microperfusion with
GABAA antagonists, bicuculline, or picrotoxin in the dorsal raphe has been reported to lift inhibition of serotonergic neurons during sleep (Levine and Jacobs, 1992) and also to decrease PS (Nitz
and Siegel, 1997a). In summary, within the raphe, the inverse correlations of percent PS with GABAergic versus serotonergic c-Fos-expressing neurons suggests together with other evidence, that
GABAergic raphe neurons are PS-on cells, which may be partly responsible for the important inhibition of codistributed serotonergic PS-off cells.
In the locus coeruleus and subcoeruleus, there was a significant
decrease in the numbers of noradrenergic c-Fos-expressing cells
during PS recovery as compared with deprivation and a significant negative correlation in these numbers with the percent PS across conditions. These results support the claim that noradrenergic locus
coeruleus neurons cease firing during PS and may accordingly play a
permissive role in PS generation (Hobson et al., 1975; McCarley and
Hobson, 1975; Aston-Jones and Bloom, 1981a). With carbachol-induced PS
in cats, no significant difference was documented in c-Fos-expressing
noradrenergic neurons relative to controls (Yamuy et al., 1995). In the
present study, PS recovery resulted in an increase in the number of
c-Fos-expressing GABAergic neurons, which were located in the vicinity
of the noradrenergic cells and which could thus act as local inhibitory
neurons. These GABAergic cells could represent a proportion of the
non-noradrenergic c-Fos-expressing cells in the locus coeruleus region
that were found to be increased with carbachol-induced PS in cats
(Yamuy et al., 1995). In support of increased activity during PS of
GABAergic neurons innervating the locus coeruleus, biochemical studies
have found greater release of GABA in the locus coeruleus during PS
than during SWS or waking (Nitz and Siegel, 1997b). Moreover, the
cessation of discharge by locus coeruleus neurons during this state can
be reversed by microinjection of the GABAA antagonist
bicuculline (Gervasoni et al., 1998), which also results in a decrease
in PS (Kaur et al., 1997). In summary, the present results showing
inverse correlations of PS with GABAergic versus noradrenergic
c-Fos-expressing neurons provide evidence that GABAergic neurons
are active during PS and may be responsible for the important
inhibition of adjacent noradrenergic neurons during PS.
Interaction between cell groups
The present results substantiate the concept that cholinergic
tegmental neurons are actively involved, as PS-on cells, whereas monoaminergic neurons may be permissively involved, as PS-off cells, in
PS generation (Sakai, 1988; McCarley et al., 1995). Given
pharmacological evidence that serotonin and noradrenaline inhibit
cholinergic tegmental neurons (Luebke et al., 1992; Williams and
Reiner, 1993; Leonard and Llinas, 1994; Leonard et al., 1995), the
monoaminergic neurons could tonically inhibit cholinergic neurons
during the waking state, rendering them PS-on/Wake-off cells. The
reciprocal increase in noradrenergic locus coeruleus c-Fos-expressing
neurons and decrease in cholinergic laterodorsal tegmental
c-Fos-expressing neurons during PS deprivation could reflect such an
influence in the present study, as well as confirming the role of
noradrenergic neurons in behavioral and cortical arousal and stress
(Foote et al., 1980; Aston-Jones and Bloom, 1981a,b; Abercrombie and
Jacobs, 1987; Jones, 1991c; Pezzone et al., 1993; Tononi et al., 1994).
However, in view of the reported high levels of ACh release in the
thalamus during waking (Williams et al., 1994), it is unlikely that all
cholinergic tegmental neurons are tonically inhibited during this
state. Another possibility is that a subset of cholinergic neurons
having particular projections are PS-on/Wake-off cells, such as those
projecting into the brainstem (Jones, 1990), where ACh release is
greater during PS than during waking (Kodama et al., 1990, 1992).
Electrophysiological evidence has recently been presented that there
are two subsets of putative cholinergic neurons, one that is
PS-on/Wake-off and inhibited by serotonin, and another that is
PS-on/Wake-on and unaffected by serotonin (Thakkar et al., 1998). The
present results would support the hypothesis that a subset of
cholinergic neurons may be selectively active during PS and
reciprocally related in their activity to monoaminergic neurons across
the sleep-waking cycle.
In addition, the present results indicate that GABAergic neurons that
are codistributed with cholinergic and monoaminergic neurons in the
pontomesencephalic tegmentum are also active during PS and could
accordingly, as PS-on cells, inhibit the monoaminergic PS-off cells.
Simultaneous unit recordings of putative cholinergic PS-on and
monoaminergic PS-off cells during transitions into and out of PS have
revealed mirror image changes in their discharge, such as to suggest a
mutual inhibitory relationship between them (Sakai, 1988). Because ACh
is excitatory to monoaminergic neurons (Egan and North, 1986; Li et
al., 1998), the cholinergic cells could only effect such an inhibition
via local interneurons (Jones, 1991b). Reported putative noncholinergic
PS-on cells that are excited by carbachol (Sakai and Koyama, 1996)
could correspond in part to such GABAergic interneurons. GABAergic
neurons in the pontomesencephalic tegmentum would accordingly play an
integral role in the generation of PS.
| |
FOOTNOTES |
Received Oct. 19, 1998; revised Jan. 22, 1999; accepted Feb. 1, 1999.
This research was supported by a grant to B.E.J. from the Medical
Research Council of Canada. We would like to thank Melodee Mograss for
her contribution to pilot studies.
Correspondence should be addressed to Dr. Barbara E. Jones, Montreal
Neurological Institute, 3801 University Street, Montreal, Quebec H3A
2B4, Canada.
| |
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ABSTRACT
INTRODUCTION
