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The Journal of Neuroscience, November 15, 2000, 20(22):8452-8461
Neurotensin-Induced Bursting of Cholinergic Basal Forebrain
Neurons Promotes  and  Cortical Activity Together with Waking and
Paradoxical Sleep
Edmund G.
Cape,
Ian D.
Manns,
Angel
Alonso,
Alain
Beaudet, and
Barbara E.
Jones
Department of Neurology and Neurosurgery, McGill University,
Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4
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ABSTRACT |
Cholinergic basal forebrain neurons have long been thought to play
an important role in cortical activation and behavioral state, yet the
precise way in which they influence these processes has yet to be fully
understood. Here, we have examined the effects on the
electroencephalogram (EEG) and sleep-wake state of basal forebrain
administration of neurotensin (NT), a neuropeptide that has been shown
in vitro to potently and selectively modulate the cholinergic cells. Microinjection of (0.1-3.0 mM) NT into
the basal forebrain of freely moving, naturally waking-sleeping rats produced a dose-dependent decrease in  (~1-4 Hz) and increase in
both  (~4-9 Hz) and high-frequency  activity (30-60 Hz)
across cortical, areas with no increase in the electromyogram. These EEG changes were accompanied by concomitant decreases in slow wave
sleep (SWS) and transitional SWS (tSWS), increases in wake, and most
remarkably, increases in paradoxical sleep (PS) and transitional PS
(tPS), despite the virtual absence of SWS. The effects were attributed
to direct action on cholinergic neurons as evidenced by selective
internalization of a fluorescent ligand, Fluo-NT, in choline
acetyltransferase (ChAT)-immunoreactive cells and stimulation by NT of
bursting discharge in juxtacellularly recorded, Neurobiotin-labeled, ChAT-immunoreactive neurons. We conclude that NT-induced rhythmic bursting of cholinergic basal forebrain neurons stimulates rhythmic oscillations and across the cerebral cortex. With the selective action of NT on the cholinergic cells, their bursting discharge promotes and independent of motor activity and thereby also stimulates and enhances PS.
Key words:
acetylcholine; choline acetyltransferase; EEG; sleep-wake states; slow wave sleep;
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INTRODUCTION |
As the primary source of cholinergic
innervation to the cerebral cortex (Lehmann et al., 1980 ; Rye et al.,
1984), basal forebrain cholinergic neurons have been shown to play an
important role in the modulation of cortical activity with behavioral
state. Although lesions of the basal forebrain cause a decrease in
cortical acetylcholine (ACh) release and cortical activation (LoConte
et al., 1982), stimulation causes an increase in cortical ACh release and cortical activation (Casamenti et al., 1986). During the natural sleep-waking cycle, ACh release is higher during waking and
paradoxical sleep (PS), when cortical activation occurs, than during
slow wave sleep (SWS) (Celesia and Jasper, 1966; Jasper and Tessier, 1971).
In the study of the discharge patterns of the cholinergic basal
forebrain neurons that would determine the way in which they modulate
the cerebral cortex, it was recently found that identified cholinergic
cells discharge in vivo in high-frequency bursts (Manns et
al., 2000), in a manner similar to that originally described in
vitro and shown to be dependent on intrinsic calcium currents (Khateb et al., 1992). The bursting discharge in the anesthetized animal occurs rhythmically in association with cortical activation and
cross-correlated with cortical rhythmic -like activity. Such rhythmic discharge must depend on afferent input that would promote intrinsic rhythmic activity in the cholinergic cells. Indeed, NMDA,
which was shown to induce rhythmic bursting in basal forebrain cholinergic cells in vitro (Khateb et al., 1995), was found
when administered in vivo to freely moving rats to stimulate
enhanced (4-9 Hz) and (30-60 Hz) electroencephalogram (EEG)
activity together with an active waking state (Cape and Jones, 2000).
However, NMDA is not selective in its action on cholinergic cells and
would excite other noncholinergic, including GABAergic, cortically and caudally projecting neurons (Gritti et al., 1994, 1997).
Contained in brainstem and forebrain afferents to the basal forebrain
(Morin et al., 1996; Morin and Beaudet, 1998), neurotensin (NT) appears
to be unique in its capacity to modulate cholinergic basal forebrain
neurons in a highly selective manner. In autoradiographic binding
studies, the high-affinity receptor for NT (NT1) was found to be
present in 95% of cholinergic (acetylcholinesterase reactive) neurons
and not in noncholinergic neurons (Szigethy and Beaudet, 1987; Szigethy
et al., 1990). Furthermore, a fluorescent analog of NT, Fluo-NT, was
found to be internalized in a receptor-dependent manner within choline
acetyltransferase (ChAT)-immunoreactive neurons (Alonso et al., 1994;
Faure et al., 1995). In the same brain slices, NT was found to excite
cholinergic, and not noncholinergic, neurons and to stimulate the
cholinergic neurons to discharge in rhythmic bursts (Alonso et al.,
1994).
In the present study, we administered NT by local microinjection into
the basal forebrain of freely moving, naturally waking-sleeping rats
to examine the effect of NT's excitation of cholinergic neurons on
cortical activity and sleep-wake state. We also examined whether in
the same animals a fluorescent analog of the peptide, Fluo-NT, was
internalized by ChAT-immunoreactive neurons, and whether in urethane-anesthetized animals NT induced a bursting discharge in
juxtacellularly recorded and Neurobiotin (Nb)-labeled
ChAT-immunoreactive cells.
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MATERIALS AND METHODS |
Animals and surgery. Microinjections were performed
in 22 male Wistar rats (200-215 gm, Charles River, St. Constant,
Quebec, Canada) or when combined with single unit recording (see
below) in seven male Long-Evans rats (200-250 gm, Charles River). All procedures were performed in accordance with the McGill University Animal Care Committee and the Canadian Council on Animal Care.
Chronic experiments involving microinjections of NT or Fluo-NT in
freely moving animals were performed in 16 (of the 22) Wistar rats. The
rats were operated under barbiturate anesthesia (Somnotol, 67 mg/kg,
i.p.) for stereotaxic implantation of indwelling guide cannulae and EEG
and electromyogram (EMG) electrodes, as described previously (Cape and
Jones, 1998). Field potential recordings were also performed in some
animals (4 of the 16 that were used in the main and dose-response
studies below) with deep electrodes in the hippocampus [ 4.0 mm
anteroposterior (AP), 2.2 mm lateral (L), 3.5 mm ventral (V) relative
to bregma] and entorhinal cortex (7.6 mm AP, 5.2 mm L, 7.2 mm V
relative to bregma). Rats were maintained in recording chambers on a 12 hr light/dark cycle with food and water available ad
libitum.
Acute experiments involving microinjections of Fluo-NT were performed
in anesthetized animals (Somnotol, 67 mg/kg, i.p., using 6 of the 22 Wistar rats; see below). Those involving unit recording with
microinjections were also performed in anesthetized animals (urethane,
1.4 gm/kg, i.p., using seven Long-Evans rats; see below).
Microinjections of NT. In chronic experiments involving
microinjections of NT (in 12 of the 16 rats), the Ringer's and
NT microinjections were performed in several stages (see Fig. 1) by use
of a remotely controlled device as described previously (Cape and
Jones, 1998). They were performed bilaterally in a volume of 0.5 µl
solution per side and were delivered using 1 µl syringes, which were
driven simultaneously by one syringe pump. In pilot experiments,
previously published concentrations (1.0, 2.0, and 3.0 mM)
of NT (Faure et al., 1995) were tested (in 3 of the 12 rats) before
selection of 1 mM concentration of NT that was used for the
main series of experiments (in 11 rats, including the 3 used in pilot
experiments plus 8 other rats). Complete dose-response series were
performed (in four rats, including three of the previous eight from the
main series plus one other rat) by repeated trials using concentrations
of 0 (Ringer's), 0.10, 0.25, 1.00, and 3.00 mM. Doses were
administered in randomized order and separated by a minimum interval of
48 hr. Antagonism of the NT effect was tested in repeated trials (in
three of the previous four rats in dose-response series) using
atropine (30 mg/kg, i.p.) that was administered at the time of
insertion of the inner cannulae, at ~30 min before the microinjection
of (0.25 mM) NT.
Microinjection of Fluo-NT. Fluo-NT was administered in
chronic experiments at a concentration of 0.25 mM in the
same way as NT (above) in (4 of the 16) freely moving rats. Acute pilot
experiments were performed (in three of the six) anesthetized rats
(Somnotol, 67 mg/kg, i.p.) to test different concentrations (1.0, 0.50, and 0.10 mM) of Fluo-NT for the adequacy of fluorescent
labeling. The fluoresceinylated derivative of NT, Fluo-NT, was kindly
supplied by Advanced Bioconcept (Montreal, Quebec, Canada). It was
dissolved together with a peptidase inhibitor, kelatorphan (15 mg/ml),
in Ringer's solution.
The specificity of the Fluo-NT labeling was tested in acute experiments
in three of the six anesthetized animals (Somnotol, 67 mg/kg, i.p.) by
administration of (0.22 mM) Fluo-NT bilaterally and excess
unlabeled (1.3 mM) NT unilaterally.
EEG recording and analysis. In the chronic experiments
(n = 16), signals were recorded using a Grass model 78D
polygraph and sent to a computer for digitization, storage, and
subsequent analysis using Stellate Systems software (Montreal, Quebec).
Behaviors were simultaneously observed using a video camera and scored
on the record as annotations according to set categories, as described previously (Maloney et al., 1997; Cape and Jones, 1998). Recording was
performed before and after microinjections during the period of
~11:00 A.M. to 3:00 P.M.
Each record was scored off-line by visual assessment for classification
of 20 sec epochs according to sleep-wake state. The EEG and EMG
activities were considered together with the behavioral annotations for
scoring as wake (W), transition into SWS (tSWS), SWS, transition into
PS (tPS) or PS (Maloney et al., 1997). Spectra were computed using
Stellate Systems software by fast Fourier transform (FFT) based on 512 points for 2 sec epochs (or 256 samples per second) with a resolution
of 0.5 Hz and a range of 1.5-63.5 Hz. Frequency bands were set as (1.5-4.0 Hz), (4.5-8.5 Hz),  (9.0-14.0 Hz),  1 (14.5-18.5
Hz), 2 (19:0-30.0 Hz), and (30.5-58.0 Hz). On the basis of
visually scored records, sleep-wake hypnograms were displayed in
association with EEG frequency band activities for 20 sec epochs and
data acquired from these files for the 30 min post-injection period
using Eclipse software (Stellate Systems). Spectral analysis was also
performed on 4 sec epochs for analysis of peak frequencies in
association with band amplitudes using Rhythm software (Stellate
Systems). Spectrum and frequency band activities were displayed and
reported in analog-to-digital (A/D) converted amplitude units. The
ratio of / was calculated and reported as a measure of activity in the EEG (Maloney et al., 1997). EMG amplitude was computed
for the total spectrum up to 58.0 Hz. The EEG and EMG data for the
figures were plotted with Origin (v5.0, Microcal Software, Northampton, MA).
Based on examination of EEG records and hypnograms from all animals,
data from 2 of 16 rats in the chronic series were eliminated from the
study because of the presence of seizure activity that is not uncommon
in Wistar rats and emerged here after NT microinjections. These two
rats had been used in the main series of NT microinjections (n = 11, thus reduced to 9). For all other rats,
average EEG and state data for the post-injection period were analyzed
by paired t test or ANOVA using repeated measures (or
grouping by rat) depending on the specific experiment, as specified in
Tables and Figure legends. Statistical comparisons of , , and
/ EEG activity and EMG activity, which have been shown to vary
significantly as a function of behavior and state (Maloney et al.,
1997), were performed. All statistics were performed using Systat
(v9.0) software (Evanston, IL).
Unit recording and labeling. For combined unit recording and
microinjections, acute experiments were performed on seven male Long-Evans rats, which were anesthetized (urethane, 1.4 gm/kg) and
held in a stereotaxic frame. Microinjections in these experiments were
performed unilaterally on the same side as the single unit recording.
Adequate anesthesia was confirmed by the lack of hindlimb withdrawal in
response to pinch, and additional anesthetic (0.1-0.15 gm/kg) was
administered if withdrawal occurred. Body temperature was
thermostatically maintained above ~35°C with a heating pad. Juxtacellular recording and labeling of cells with Nb (Vector Laboratories, Burlingame, CA) was performed using an intracellular amplifier (IR-283; Neuro Data Instruments, New York, NY) as described previously (Pinault, 1996; Manns et al., 2000). EEG was also recorded from retrosplenial cortex and prefrontal cortex. Unit discharge was
examined before, immediately after, and during the maximal effect of NT
microinjection. Using 1 min epochs of stationary data, the average
discharge rate was calculated from the peristimulus histogram (PSH),
the predominant instantaneous firing frequency was determined from the
first-order interspike interval histogram (ISIH), and the presence of
rhythmic unit and unit-to-EEG cross-correlated discharge was assessed
by autocorrelation histogram (ACH) and spike-triggered averaging
(STA), as described previously (Manns et al., 2000). On the basis of
the properties of unit discharge and the immunostaining of the
Nb-labeled cells for ChAT (see below), data from cholinergic cells
(n = 3) were selected for detailed analysis and presentation.
Immunohistochemistry. At the conclusion of the chronic
experiments, animals were killed with a lethal dose of pentobarbital (Somnotol, ~120 mg/kg, i.p.) and perfused through the heart with a
fixative solution [3.0% paraformaldehyde, as published previously (Gritti et al., 1997)]. In both chronic and acute experiments involving microinjections of Fluo-NT, animals were killed 15 min after
the microinjection by perfusion. In acute experiments involving unit
recording and microinjection, the urethane-anesthetized animals were
killed by perfusion after completion of the microinjection and
subsequent juxtacellular labeling of the recorded unit.
In animals having received microinjections of NT, brains were processed
for ChAT immunostaining to examine the injection sites in relation to
the cholinergic cells. Immunohistochemistry was performed using the
peroxidase-antiperoxidase (PAP) technique as published previously
(Gritti et al., 1997). Sections were incubated overnight with rabbit
anti-ChAT antiserum (1:3000; Chemicon International, Temecula, CA)
followed by donkey anti-rabbit secondary antisera and rabbit PAP
antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and
revelation with diaminobenzidine. Cells were plotted using an image
analysis system (Biocom, Paris, France).
In animals having received microinjections of Fluo-NT, 20-µm-thick
frozen sections were cut and collected every 200 µm for processing.
Half of the series was cold-mounted for simple visualization of the
Fluo-NT fluorescence, and the other half was incubated with anti-ChAT
antiserum for dual visualization of Fluo-NT and ChAT. The latter
sections were free-floated at 4°C overnight in rabbit anti-ChAT
antiserum (1:200) and then at room temperature for 2 hr in goat
anti-rabbit Texas Red (1:50; Jackson).
Brains in which recorded units were labeled with Nb were processed for
dual staining of Nb and ChAT. Frozen sections were cut (30 µm) and
incubated overnight in rabbit anti-ChAT antiserum (1:3500) followed by
incubation with Cy2-conjugated streptavidin (1:800; Jackson) to reveal
Nb and with Cy3-conjugated donkey anti-rabbit antiserum (1:1000;
Jackson) to reveal ChAT. Sections were then mounted and viewed by
fluorescent microscopy using a Leitz Dialux microscope equipped with a
Ploemopak 2 reflected light fluorescence illuminator with Leica filter
cubes for fluorescein (I3) and rhodamine (N2.1).
Confocal microscopy. Sections were examined using a Zeiss
confocal laser scanning microscope (CLSM 410) equipped with an Axiovert 100 inverted microscope and an argon/krypton laser. FITC signals for
Fluo-NT were imaged by exciting samples with 488 nm light, and Texas
Red for ChAT was imaged in the same sections by 568 nm light.
Images were acquired sequentially as single transcellular optical
sections and averaged over 32 scans per frame. Images, processed using
the Carl Zeiss CLSM software (v3.1), were prepared for publication
using Adobe Photoshop (v5.0, Adobe Systems, San Jose, CA).
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RESULTS |
The microinjection cannulae were symmetrically placed above the
major population of basal forebrain cholinergic neurons as was evident
by the tracks of the cannulae seen in relation to ChAT-immunostained
cells in processed brains (Fig. 1). The
tracks were evident passing through the caudate putamen and medial part of the globus pallidus and extending ventrally into the substantia innominata (SI) above the magnocellular preoptic nucleus (MCPO), where
large numbers of ChAT-immunoreactive cells are located.
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Figure 1.
Schematic atlas section through the basal
forebrain showing the location of the microinjection cannulae (based on
visible tracks) in relationship to ChAT-immunoreactive neurons. Inner
cannulae filled with Ringer's or NT were first inserted into
indwelling guide cannulae (to within ~2 mm of tip as indicated),
where they were held until the time of injection. Immediately before
injection, inner cannulae on both sides were lowered by a remote
driving mechanism to pass out of the guide cannulae to the injection
site (~2 mm below guide cannulae as indicated) in the SI and above
the MCPO. The representation of the injected fluid is based on
estimates previously established with neuroanatomical tracers. In this
area and beyond within the SI and MCPO, punctate fluorescent labeling
was visible with injections of Fluo-NT within ChAT-immunoreactive cells
(see Fig. 7A-D). Also within the SI and
MCPO, cells (stars on left side) that
were juxtacellularly recorded and labeled with Nb during NT
microinjections in anesthetized animals were identified as
ChAT-immunoreactive (see Fig.
7E-H). ac,
Anterior commissure; CPu, caudate putamen;
GP, globus pallidus; MCPO, magnocellular
preoptic nucleus; SI, substantia innominata.
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Effects of NT microinjections on EEG and sleep-wake states
Microinjections of (1.0 mM) NT into the basal
forebrain altered the EEG and natural sleep-waking cycle of the rat
(n = 9) (Fig. 2). From
the moment the NT-filled cannulae were lowered into the tissue and
through the injection and post-injection periods (Fig.
2B), activity was diminished and associated with
a loss of SWS during the day, when rats are normally in SWS the
majority of the time (Fig. 2A). In the absence of activity, activity was relatively high and associated with moderate
activity. As judged from the EEG, EMG, and behavioral observations
used in the visual scoring of sleep-wake states, a state of PS or tPS occurred in the absence of SWS and alternated directly with a state of
wake. From the EMG amplitude, which usually remained low, it appeared
that the animals remained quiet while awake as well as asleep after the
NT. As confirmed in the behavioral annotations, the wake state after NT
was predominantly quiet and characterized by a reclining, outstretched
posture with head down. The passage into tPS or PS was usually marked
only by closing of the eyes, with no other postural change. At one
moment, the rat would be lying quietly with eyes open showing "quiet
waking" behavior, and the next moment, without changing position, it
would close its eyes and begin twitching, thus showing "sleeping with
twitches" or PS behavior. All rats receiving (1.0 mM) NT (9 of 9) showed transitions from wake
directly into PS or tPS with no intervening SWS or tSWS. Active
behaviors, including moving and eating, which were accompanied by high
EMG, were also seen occasionally after NT, because they occur normally
during this period of the day (Maloney et al., 1997). A direct passage
from these active or attentive waking behaviors to a PS-like state was
never seen. None of the behaviors or postures appeared abnormal.
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Figure 2.
Hypnograms showing sleep-wake state changes in
conjunction with EEG and EMG activity changes before and after
injection of Ringer's (left) and NT
(right, rat B11). After insertion of the Ringer's- or
NT-filled cannulae into the indwelling cannulae (Fig. 1) and resumption
of sleeping by the animal, recording was resumed, and the filled
cannulae were lowered into the tissue for injection ~2 min later that
lasted ~5 min (Fig. 1). Note that during and after the entire
microinjection procedure, Ringer's does not appear to disrupt the
sleep-wake cycle and associated EEG activity. In contrast, NT
markedly changes the cycle and EEG from the moment the filled
cannulae are lowered into the tissue and for the duration of the
injection and post-injection period. After NT, is diminished, and
SWS is absent. becomes relatively high and together with increases in association with the occurrence of PS directly after
waking in the absence of SWS. Sleep-wake states were scored off-line
by visual assessment of the record together with behavioral
annotations. The EEG is from the right retrosplenial lead and shows
band activities for (30-58 Hz), (1.5-4 Hz), and (/:
4.5-8.5 Hz/1.5-4 Hz); EMG is from the neck muscles (1-58 Hz). Each
activity is scaled to maximum and reported in AD units (where for EEG
100 µV  125 AD units) for 20 sec epochs.
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Across animals receiving (1.0 mM) NT, there was a
significant decrease in relative band activity and a significant
increase in relative band activity and / ratio as compared
with Ringer's on the retrosplenial lead (n = 9) (Table
1). There was no significant change in
EMG. Sleep-wake states were significantly altered in amount, with a
significant decrease in SWS, significant increase in wake, and
significant increase in PS and tPS. In one animal (B29), PS reached
23% and tPS reached 36%, representing together ~60% of total
recording time. Within the individual states (except SWS because of the
few epochs after NT), relative and , as well as / ratio,
and EMG measures were not significantly different between the Ringer's
and NT conditions across animals (for the 9 rats presented in Table 1,
p > 0.05, according to paired t tests per
state; data not shown). Varying according to behavior, wake epochs
could be characterized by moderate with high EMG activity
(associated with active waking) after Ringer's and NT, but they were
also and most often characterized by moderate with low EMG activity
(associated with quiet waking) after NT (Fig.
3; and see below). SWS was absent in most
animals after NT and replaced predominantly by tPS leading to PS (Fig.
3; and below). As a transitional state (Maloney et al., 1997), tPS
varied between a predominance of spindle activity accompanied by ,
which was most characteristic after Ringer's, to a predominance of accompanied by spindle activity, which was most characteristic after NT
(Fig. 3; and below). PS was similar in the two conditions (Fig. 3). As
in baseline conditions (Maloney et al., 1997), EMG after Ringer's and
NT was low throughout sleep [when the electrocardiogram (ECG) signal
also becomes evident] (Fig. 3) and often slightly higher in PS because
of the muscular twitches of whiskers, ears, and body musculature that
are picked up on the electrodes in the neck muscles.
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Table 1.
Relative EEG activity and time spent in sleep-wake states
during the 30 min post-injection period after Ringer's and (1 mM) NTa
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Figure 3.
Sample recordings of EEG from retrosplenial cortex
and EMG from neck muscles during states observed after
Ringer (top) and NT
(bottom). EMG also shows ECG activity when postural
muscle tonus is low. Note the presence of activity with active
waking after Ringer's and with quiet waking after NT (as evident by
respective EMG signals), the absence of SWS after NT, the occurrence of
tPS with a predominance of after NT, and the similar appearance of
PS in both conditions. (Amplitudes shown as AD units are presented in
Fig. 2.)
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Although the predominant EEG activity after Ringer's was composed of
activity (1.5-4 Hz) in association with SWS (Fig.
4), the predominant EEG activity after NT
was composed of -like activity (4.5-8.5 Hz) in association with
wake, tPS, or PS (Fig.
5A-C) on all
cortical leads. In comparison with the Ringer's SWS activity, the
NT -like activity was consistently associated with higher activity (30-58 Hz) (Figs. 3, 4). The EMG was low, indicative of
postural muscle relaxation or atonia during the quiet waking, tPS, and
PS states (Fig. 5). In spectra from 4 sec epochs sampled during the
post-injection period (~4-12 min) during which the maximal, stable
effect of NT was observed, the average low peak frequency was
significantly higher across cortical areas after NT (n = 5) (Table 2). The average peak
frequency shifted from the range into the range, although it
differed in specific frequency across areas, being the highest on the
retrosplenial and lowest on the frontal lead (Table 2). The differences
in peak frequencies across areas appeared to be greatest during waking behaviors and tPS and the least during PS and SWS. In animals with deep
electrodes in the hippocampus and entorhinal cortex (n = 4), activity was often evident on these leads when it was present
on other cortical leads but would also differ in frequency and phase
from that on the other cortical leads at particular moments of the
different behavioral and sleep-wake states. There was no specific peak
or shift in peak frequency in the band with NT, only an increase in
the average amplitude of the entire band (Table 2).
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Figure 4.
EEG record and spectral analysis after Ringer's
microinjection (in rat B11). SWS was the predominant state after
Ringer's and, like normal SWS, was characterized by a curled sleeping
posture behaviorally and by high amplitude activity (evident in
top, unfiltered black traces) and
relatively low amplitude activity (evident in
bottom, gray traces filtered for 30-58
Hz). These characteristics are evident in the spectra from the same 4 sec epochs (right), in which the predominant peak on all
leads is in the band (2.0 Hz) and accompanied by a relatively low
amplitude in the band. The EEG was recorded by reference to an
electrode in the rostral frontal bone from the anterior medial frontal,
retrosplenial, parietal, and occipital cortical regions (shown here for
the right side). Voltage scales are the same for all cortical leads.
(Note that the amplitude of the frontal lead is the lowest because of
its proximity to the reference electrode.) EMG recorded from the neck
muscles also shows ECG activity when postural muscle tonus is low, as
here during sleep. Spectra are displayed in amplitude
(AD) units (per 0.5 Hz; 100 µV 125 AD units). ,
, and frequency bands are differentially shaded
(as in the hypnograms in Fig. 2); and bands are
unshaded.
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Figure 5.
EEG records and spectral analyses after NT during
waking (top), tPS (middle), and PS
(bottom, in rat B11). The predominant state was wake and
often characterized behaviorally (as during this epoch shown on
top) by a quiet state and a reclining posture. The
unfiltered EEG is dominated by the occurrence of rhythmic slow activity
on all leads that corresponds to peaks in the band (~7.0-7.5 Hz)
and moderately high amplitude activity. EMG was low, indicating
postural muscle relaxation. A large percentage of time was also spent in tPS, which often
occurred directly after wake (as in the epoch shown in the
middle), with the simple closing of the eyes while the
rat remained in a reclining, uncurled posture. In this and similar
epochs of tPS, the EEG is characterized by the presence of -like
activity mixed with slower or spindle-like activity. The spectra
reveal peaks at the border between and bands (~3.5-4.5 Hz)
on frontal, retrosplenial, and occipital leads together with a
secondary peak in the band (~10 Hz) reflecting spindle-like
activity, and a peak in the middle of the band (6.0 Hz) on the
parietal lead. band activity is high relative to SWS (Fig. 4). EMG
indicates postural muscle atonia. Typical PS, which was behaviorally
and electroencephalographically indistinguishable from normal PS,
occurred a large percentage of the time after NT. In these cases (as
for the epoch shown at the bottom), muscle twitches were
evident while the animal was in a reclining, partially curled (or also
fully curled) posture with eyes closed. The EMG shows evidence of
small twitches during postural muscle atonia (when ECG signal is also
evident). The unfiltered EEG is dominated by -like activity on all
leads, with spectral peaks at the high end of the band (~8.0 Hz)
and relatively high amplitude activity, as compared with Ringer's
SWS (Fig. 3). See Figure 4 for details.
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Table 2.
EEG frequency peaks and band amplitudes obtained from
spectral analysis of epochs from four cortical areas during the
maximal, stable effect after (1.0 mM) NT as compared with
Ringer'sa
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Increasing doses of NT were associated with increasing amplitude
and / ratio and decreasing amplitude with no systematic change in EMG (n = 4) (Fig.
6A). Paralleling the
EEG changes were dose-dependent increases in wake and in PS, tPS and
decreases in SWS, tSWS (Fig. 6B). PS and tPS
increased in a linear manner up to a maximum with (1.0 mM) NT and represented together ~40% of
recording time on average with that dose. Wake reached a maximum with
(3.0 mM) NT, representing ~75% of recording
time, at which dose, PS and tPS were similar in amount to Ringer's
(<20%).
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Figure 6.
Changes in EEG activities (left)
and sleep-wake state (right) as a function of the dose
of NT. With increasing doses, and increased and activity
decreased (left). and activities, as well as
EMG, are presented, as average amplitude in AD units (100 µV 125 AD units) and as the ratio of / for 20 sec epochs during the
30 min post-injection period (mean ± SEM from 4 rats). Statistics
were performed on natural log values that were normally distributed.
The data were analyzed by ANOVA with dose as a repeated measure
(performed with the metric 0, 0.1, 0.25, 1.0, and 3.0 mM)
and post hoc polynomial contrasts for linear trend
analysis (showing a significant trend for , according to the cubic
polynomial: F = 10.45, df = 1,3, p = 0.048; for , according to the linear:
F = 47.25, df = 1,3, p = 0.006; and for /, according to the quadratic:
F = 38.76, df = 1,3, p = 0.008; *p < 0.05; **p  0.01). Although wake and PS together with tPS increased as a function
of dose, SWS together with tSWS decreased as a function of dose
(right). State data (mean ± SEM from 4 rats) are
presented as the percentage of the 30 min post-injection recording
period. In an overall test, in which dose was examined across states
(entered as a grouping factor with 5 levels), percentage state varied
significantly as a function of dose (F = 3.842, df = 4,60, p = 0.008), with a significant
interaction of state and dose (F = 4.121, df = 16,60, p = 0.000). Given the parallel changes in
transitional states, subsequent ANOVAs were performed for W, for tSWS
together with SWS, and for tPS together with PS (with state as a
grouping factor). For all states, there was a significant main effect
of dose, although the maximal trend, examined by polynomial contrasts,
differed per state (wake with the linear polynomial:
F = 54.666, df = 1,3, p = 0.005; tSWS, SWS with the linear: F = 29.057, df = 1,6, p = 0.002; and tPS, PS with the
quadratic: F = 24.073, df = 1,6, 0.003;
**p 0.01).
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The changes in EEG induced by (0.25 mM) NT were largely
antagonized with the systemic administration of atropine (30 mg/kg, i.p.) (n = 3) (Table 3).
Microinjection of NT after atropine no longer produced an increase in
or decrease in as it did before atropine (Table 3). The average
/ ratio was not increased by NT after atropine; however, the
difference in the ratio between NT on the one hand and atropine and NT
together with Ringer's on the other did not reach significance, and
thus the statistical test of the hypothesis that atropine completely
antagonized the NT-induced increase in activity was not confirmed
(Table 3). Atropine by itself, however, produced a continuous waking
state during which, as also seen after the subsequent microinjection of
NT, movement could occur and be accompanied by , even though activity was predominant. Nevertheless, atropine clearly prevented the
NT-induced enhancement of tPS and PS during which was most prominent.
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Table 3.
EEG and state effects during the 30 min post-injection
period after (0.25 mM) NT after previous systemic
administration of atropinea
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Labeling of cholinergic cells after Fluo-NT microinjections
To visualize NT's target cells, the fluorescent ligand, (0.25 mM) Fluo-NT, was administered bilaterally by microinjection to the freely moving, naturally waking-sleeping rats in the same manner as the nonfluorescent ligand (Fig. 1), except that its effects
were examined for a post-injection period of 15 min to ensure that the
ligand would be present in adequate amounts for histofluorescent
visualization after rats were killed. In repeated trials
(n = 4), (0.25 mM) Fluo-NT
produced changes in EEG activity and sleep-wake states that did not
differ from those after (0.25 mM) unlabeled NT
during the 15 min post-injection period. Relative (21.1 ± 1.9%) and / ratio (1.75 ± 0.18) were similarly elevated, and relative (12.2 ± 1.4%) similarly decreased with the
fluorescent as compared with the nonfluorescent NT (: 19.6 ± 1.41%; /: 1.78 ± 0.19%; : 12.1 ± 1.59%). Wake
(41.7 ± 13.44%) and tPS+PS (28.9 ± 9.59%) were also as
high after Fluo-NT, as after unlabeled NT (wake: 37.22 ± 11.45%;
tPS+PS: 28.9 ± 11.33%).
In brains of animals having received microinjections of Fluo-NT
(n = 4), evidence of fluorescent labeling was visible
by light microscopy. A diffuse fluorescent staining of the cytoplasm of a small number of cells was evident in the immediate vicinity of the
injection tracks. Beyond this region, very light and fine labeling was
detectable in cells within the SI and MCPO. When examined by confocal
laser scanning microscopy, this fine fluorescent staining was seen to
correspond to punctate labeling within the cytoplasm of neuronal
perikarya (Fig.
7A,C).
The punctate staining was evident predominantly in medium to large
cells, which varied in shape from fusiform to polygonal. Such Fluo-NT+
cells were found within the SI and MCPO extending in a radius of up to
1 mm from the injection tracks and thus beyond the region that was estimated by volume of the solution as the immediate injection site
(Fig. 1). In sections immunostained for ChAT, it appeared that although
diffuse fluorescent labeling was present in ChAT-negative cells in the
immediate vicinity of the tracks, the Fluo-NT punctate labeling was
found within ChAT-positive neurons in the SI and MCPO (Fig.
7B,D).
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Figure 7.
Photomicrographs showing fluorescent staining
after Fluo-NT microinjections (A-D) and Nb
juxtacellular application (E, F)
in association with ChAT-immunostaining. When viewed under illumination
for fluorescein (A, C), punctate
fluorescent granules (arrowheads) were visible in the
cytoplasm of perikarya and proximal dendrites of relatively large
neurons, which when viewed under illumination for rhodamine were found
to correspond to ChAT-immunoreactive cells (B,
D). Neurons juxtacellularly recorded and labeled with Nb
(revealed by green fluorescent Cy2-conjugated streptavidin) in the SI
and MCPO (E and F, respectively;
indicated by stars in Fig. 1) were identified as
ChAT-immunoreactive (G and H; revealed by
red fluorescent Cy3-conjugated secondary antibodies). Scale bar, 20 µm.
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In acute experiments in anesthetized rats (n = 3) that
were aimed at testing the specificity of the Fluo-NT labeling, it was found that although diffuse labeling of neurons was still visible around the injection track, punctate Fluo-NT perikaryal labeling was no
longer visible in neurons in the SI and MCPO on the side where excess
unlabeled NT was injected in combination with the (0.22 mM) Fluo-NT.
Effects of NT microinjections on the discharge of
cholinergic neurons
The effect of NT microinjection after the discharge of basal
forebrain cholinergic units was examined in urethane-anesthetized animals. Each unit was labeled by juxtacellular application of Nb for
subsequent histochemical processing and examination of ChAT
immunoreactivity. Three units displayed discharge properties typical of
cholinergic neurons according to previously established characteristics, notably the distinguished presence of rhythmic high-frequency burst discharge with somatic stimulation-induced cortical activation (Manns et al., 2000). Of these, two were
successfully labeled with Nb (Fig.
7E,G), and both of these were found
to be positive for ChAT immunostaining (Fig.
7F,H). One was located in
the SI and the other in the MCPO (Fig. 1). These Nb+/ChAT+ neurons, as
well as the electrophysiologically characterized cholinergic-like cell,
all showed changes in their rate and pattern of discharge that were
associated with changes in EEG activity after the NT microinjection as
compared with the pre-injection recording baseline (Fig.
8). All increased their average rate of
discharge (according to PSH measures) and instantaneous firing
frequency (according to ISIH measures) shortly after, if not before,
the end of the NT microinjection (Fig. 8, middle
panel). Additionally, they all showed high frequency bursts
(>80 Hz) in their activity after NT that were infrequent preceding NT
in the unstimulated condition (Fig. 8, arrows). In two
cells, this bursting discharge became rhythmic spontaneously (Fig. 8,
SI; and data not shown), and in the other cell, it did so
with tail pinch (Fig. 8, MCPO, as evaluated by
autocorrelation histogram measures) in parallel with the appearance of
rhythmic slow activity on the EEG. The rhythmic burst discharge of all
three was temporally cross-correlated with the retrosplenial and
prefrontal EEG signals [as evaluated by STA, see Manns et al.
(2000)]. The rhythmic discharge of the MCPO unit matched the predominant activity in the retrosplenial cortex, whereas that of the
SI unit matched the slower predominant activity in the prefrontal
cortex (as evident in spectral analysis).
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Figure 8.
Discharge patterns of Nb+/ChAT+ neurons in SI
(top; cell in Fig.
7E,F) and MCPO
(bottom; cell in Fig.
7G,H) in association with NT
unilateral microinjection in urethane-anesthetized animals. Presented
are EEG activity from prefrontal (PFCx) and
retrosplenial cortex (RSCx) and associated unit activity
before, 1 min after, and several minutes after the NT microinjection.
Note the change from a single spiking discharge to a burst discharge,
in addition to an increased rate of discharge, after NT microinjection.
This burst discharge takes on a rhythmic character in association with
EEG activation after NT (spontaneously in SI unit and with somatic
stimulation in MCPO unit). Arrows indicate examples of
high-frequency bursts (>80 Hz and in some cases beyond resolution of
the printing).
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DISCUSSION |
The results of the present study demonstrate that NT, which
selectively binds to, internalizes within, and induces bursting of
cholinergic basal forebrain neurons after local microinjection, diminishes activity with SWS and stimulates and activity with wake and PS states.
Visualization of the fluorescent analog, Fluo-NT, and recording of unit
activity in ChAT-immunoreactive neurons both indicated here that NT
injected into the basal forebrain targeted and directly altered the
discharge of the cholinergic cells therein. After its microinjection,
Fluo-NT was found to be selectively internalized within
ChAT-immunoreactive neurons, reflecting its selective high-affinity binding to those cells (Faure et al., 1995). Moreover, juxtacellularly recorded and Nb-labeled, ChAT-immunoreactive neurons were found to
increase their rate of firing and to discharge in bursts after the
microinjection of NT here in anesthetized animals, similarly to the
manner in which identified cholinergic neurons were shown to be
directly modulated by NT in the slice (Alonso et al., 1994).
In the naturally waking-sleeping animals, NT microinjections promoted
cortical activation, which was evident as a decrease in and an
increase in activity during the day when the rats are normally in
SWS the majority of the time. These EEG changes may be attributed to
the release of ACh from terminals of the discharging cholinergic
basalocortical neurons and postsynaptic action of ACh on cells in the
cerebral cortex. ACh has been shown to depolarize pyramidal cells
through long-acting muscarinic receptors that close
K+ channels and thus increase excitability
and promote tonic firing in these cells (Krnjevic, 1967; McCormick and
Prince, 1986; Metherate et al., 1992). Here, the increase in was
antagonized by previous administration of atropine, the muscarinic
antagonist, confirming the importance of cortical ACh release and
postsynaptic action on muscarinic receptors for this EEG change after
the NT injections.
In addition to an increase in activity, there was a marked increase
in activity after NT microinjections into the basal forebrain.
Induction of activity was also shown to occur in another study
using intracerebroventricular administration of NT (Castel et al.,
1989). The promotion of activity in both cases may be caused by the
stimulation of rhythmic burst discharge of basalocortical cholinergic
neurons and thus rhythmic release of ACh from their terminals in the
cortex. Here, as in previous studies, the precise frequency of -like
activity was found to differ across cortical leads and could not be
attributed to a volume-conducted signal from the hippocampus or
entorhinal cortex, as considered previously (Maloney et al., 1997). The
peak frequency was highest on the retrosplenial cortical lead and
lowest on the frontal lead, which is over the anterior medial or
prefrontal cortex. It was shown previously that in the
cingulate-retrosplenial cortex is generated there and persists after
lesions of the medial septum (Borst et al., 1987; Leung and Borst,
1987), thus being independent of the septohippocampal system. These
data strongly suggested that cingulate-retrosplenial is dependent
on input from the basal forebrain. Indeed, it was found recently that
identified cholinergic basal forebrain neurons discharge in rhythmic
bursts in association with stimulation-induced -like activity from
the retrosplenial and also prefrontal cortex in urethane-anesthetized rats (Manns et al., 2000), thus substantiating this possibility. Here, rhythmic bursting activity that was cross-correlated with rhythmic EEG activity was seen in Nb-labeled ChAT-immunoreactive neurons after NT microinjections. As had been found in the former study, it was also found in these units that the rhythmic bursting was
at a higher frequency for the unit with a discharge that matched the
predominant retrosplenial activity than for the unit with a discharge
that matched the slower predominant prefrontal activity. These results
suggest that bursting cholinergic neurons modulate cortical activity in
a -like manner, with potentially different frequencies for different
cortical areas during some behaviors yet with the possibility for
coordinated discharge at similar frequencies during other behaviors and
states. Accordingly, the activity measured on different cortical
leads after NT microinjections in the freely moving, naturally
waking-sleeping rats may be stimulated by rhythmically bursting
cholinergic neurons projecting to different regions of the cerebral
cortex and stimulating regionally particular or coordinated frequencies
of -like activity during the resulting behavioral states.
In contrast to the changes in and , the increase in the /
ratio after NT microinjections was attenuated but not conclusively antagonized by atropine, because the change was not statistically significant. As noted here, however, atropine produces a dissociated state of behavioral waking with slow EEG activity, yet sometimes with
activity when animals are moving (Vanderwolf, 1975). on the EEG
after NT microinjections following atropine could represent such
atropine-resistant occurring during active waking. Recent evidence
has indicated that in addition to the cholinergic neurons, a small
subset of noncholinergic neurons in the diagonal band-medial septum
are modulated by NT in vitro (Matthews, 1999). It is
possible that atropine-resistant may be stimulated by NT through
direct action on such noncholinergic neurons, although such action was not found on the noncholinergic neurons sampled in vitro in
the substantia innominata-magnocellular preoptic region (Alonso et al., 1994). in the cortex as in the hippocampus may depend on noncholinergic, in addition to cholinergic, neurons (Lee et al., 1994).
Here after NT microinjections, noncholinergic neurons could also be
activated secondarily by local projections of the cholinergic neurons
in the basal forebrain. In the presence of atropine, cholinergic transmission through local and distant projections would depend on
nicotinic receptors. Atropine-resistant in the cortex could be
stimulated by ACh released from rhythmically discharging cholinergic neurons by being transmitted through nicotinic receptors that have been
shown to be located on interneurons (Xiang et al., 1998; Porter et al.,
1999). Indeed, fast-acting, nicotinic receptors could be important in
the temporally punctuated modulation of cortical activity that must
occur with a -like frequency (Manns et al., 2000). Nonetheless,
slower depolarization through muscarinic receptors can also bring
cortical neurons within the optimal subthreshold range in which they
can oscillate at a frequency (Klink and Alonso, 1993), and faster
depolarization of interneurons through muscarinic receptors (McCormick
and Prince, 1986) could further shape the oscillations at this
frequency. Here, the most prominent that occurred with enhanced PS
and tPS after NT was fully antagonized by atropine, indicating the
importance of both the cholinergic neurons and muscarinic receptors in
this response to NT.
The changes induced by NT in cortical activity were also associated
with remarkable changes in sleep-wake states. First, SWS was
diminished in a dose-dependent manner by the NT microinjections during
the time of day when the rats are normally in SWS the majority of the
time. Wake was reciprocally increased. Yet, it was a quiet wake state
with low EMG and often marked by a reclining, normally sleeping posture
with eyes open. During this quiet waking behavioral state, activity
was present on the EEG, an association rarely if ever seen in the
normal rat (Maloney et al., 1997), in which, as documented for
hippocampal (Vanderwolf, 1975), cortical -like activity
accompanies moving or attentive behaviors. The that was stimulated
by NMDA injections into the basal forebrain was associated, as in the
normal rat, with an active moving waking state and high EMG (Cape and
Jones, 2000), but NMDA would also be associated with stimulation of
multiple cell types in the basal forebrain, thus stimulating the high
EMG activity along with cortical activation. Here, NT microinjections
were not associated with an increase in EMG activity, perhaps because
of the selective activation of the cholinergic neurons along with their
target neurons, which must also occur during natural PS with muscle
atonia. Indeed, NT microinjections lead to a dose-dependent increase in PS and the tPS in the absence of SWS. This extraordinary
induction of EEG activity associated with the occurrence and
enhancement of narcoleptic-like PS may be produced by rhythmic bursting
discharge of cholinergic neurons without the participation of those
noncholinergic basal forebrain neurons having ascending and/or
descending projections that are recruited by additional afferents
during active waking.
From the present results, NT would appear to have a potent
capacity to modulate both cortical activity and sleep-wake state through its unique action on basal forebrain cholinergic neurons. Whether NT exerts this potential in the natural state could not be
tested here because of the lack of an appropriate water-soluble antagonist of NT that could be injected into the basal forebrain. NT is
contained in basal forebrain afferents that originate in both brainstem
and forebrain (Morin and Beaudet, 1998), including neurons in the
posterior hypothalamus, from which can be elicited by electrical
stimulation (Bland and Vanderwolf, 1972). In any event, its unique
action in the basal forebrain reveals the extraordinary capacity of the
cholinergic neurons to stimulate cortical activation with rhythmic and independent of motor activity and to promote the state of PS.
It is perhaps during such cholinergically stimulated rhythmic
modulation, and thus during PS in addition to waking, that plasticity
as well as coherence may be maximized in neocortical, as in
hippocampal, networks (Huerta and Lisman, 1993; Winson, 1993; Jones,
1998; Manns et al., 2000).
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FOOTNOTES |
Received May 11, 2000; revised Aug. 23, 2000; accepted Aug. 24, 2000.
This research was supported by a grant from the Canadian Medical
Research Council to B.E.J. E.G.C. was supported as a graduate student by the Fonds de Recherche en Santé du Québec
(FRSQ), Réseau de Santé Mentale, Axe Sommeil et Vigilance,
and I.M. was supported as a graduate student by the National Science
and Engineering Research Council of Canada (NSERC). We thank Advanced Bioconcept (Montreal, Quebec, Canada) for donation of the Fluo-NT. We
thank Dominique Nouel, Thomas Stroh, and Lynda Mainville for their
contributions to this work.
Correspondence should be addressed to Dr. Barbara E. Jones, Montreal
Neurological Institute, 3801 University Street, Montreal, Quebec,
Canada H3A 2B4. E-mail: mcbj{at}musica.mcgill.ca.
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TOP
ABSTRACT
INTRODUCTION
