Dynamic changes in GABAA receptors on basal forebrain cholinergic neurons following sleep deprivation and recovery

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DOI: 10.1186/1471-2202-8-15 · Source: PubMed
Abstract
The basal forebrain (BF) cholinergic neurons play an important role in cortical activation and arousal and are active in association with cortical activation of waking and inactive in association with cortical slow wave activity of sleep. In view of findings that GABAA receptors (Rs) and inhibitory transmission undergo dynamic changes as a function of prior activity, we investigated whether the GABAARs on cholinergic cells might undergo such changes as a function of their prior activity during waking vs. sleep. In the brains of rats under sleep control (SC), sleep deprivation (SD) or sleep recovery (SR) conditions in the 3 hours prior to sacrifice, we examined immunofluorescent staining for beta2-3 subunit GABAARs on choline acetyltransferase (ChAT) immunopositive (+) cells in the magnocellular BF. In sections also stained for c-Fos, beta2-3 GABAARs were present on ChAT+ neurons which expressed c-Fos in the SD group alone and were variable or undetectable on other ChAT+ cells across groups. In dual-immunostained sections, the luminance of beta2-3 GABAARs over the membrane of ChAT+ cells was found to vary significantly across conditions and to be significantly higher in SD than SC or SR groups. We conclude that membrane GABAARs increase on cholinergic cells as a result of activity during sustained waking and reciprocally decrease as a result of inactivity during sleep. These changes in membrane GABAARs would be associated with increased GABA-mediated inhibition of cholinergic cells following prolonged waking and diminished inhibition following sleep and could thus reflect a homeostatic process regulating cholinergic cell activity and thereby indirectly cortical activity across the sleep-waking cycle.
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BMC Neuroscience
Open Access
Research article
Dynamic changes in GABA
A
receptors on basal forebrain
cholinergic neurons following sleep deprivation and recovery
Mandana Modirrousta, Lynda Mainville and Barbara E Jones*
Address: Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal,
Quebec H3A 2B4, Canada
Email: Mandana Modirrousta - mandana.modirrousta@mcgill.ca; Lynda Mainville - lynda.mainville@mcgill.ca;
Barbara E Jones* - barbara.jones@mcgill.ca
* Corresponding author
Abstract
Background: The basal forebrain (BF) cholinergic neurons play an important role in cortical
activation and arousal and are active in association with cortical activation of waking and inactive in
association with cortical slow wave activity of sleep. In view of findings that GABA
A
receptors (Rs)
and inhibitory transmission undergo dynamic changes as a function of prior activity, we investigated
whether the GABA
A
Rs on cholinergic cells might undergo such changes as a function of their prior
activity during waking vs. sleep.
Results: In the brains of rats under sleep control (SC), sleep deprivation (SD) or sleep recovery
(SR) conditions in the 3 hours prior to sacrifice, we examined immunofluorescent staining for β
2–
3
subunit GABA
A
Rs on choline acetyltransferase (ChAT) immunopositive (+) cells in the
magnocellular BF. In sections also stained for c-Fos, β
2–3
GABA
A
Rs were present on ChAT+
neurons which expressed c-Fos in the SD group alone and were variable or undetectable on other
ChAT+ cells across groups. In dual-immunostained sections, the luminance of β
2–3
GABA
A
Rs over
the membrane of ChAT+ cells was found to vary significantly across conditions and to be
significantly higher in SD than SC or SR groups.
Conclusion: We conclude that membrane GABA
A
Rs increase on cholinergic cells as a result of
activity during sustained waking and reciprocally decrease as a result of inactivity during sleep.
These changes in membrane GABA
A
Rs would be associated with increased GABA-mediated
inhibition of cholinergic cells following prolonged waking and diminished inhibition following sleep
and could thus reflect a homeostatic process regulating cholinergic cell activity and thereby
indirectly cortical activity across the sleep-waking cycle.
Background
The basal forebrain (BF) cholinergic cells play an impor-
tant role in cortical activation and arousal (see for review,
[1,2]). Recently recorded and labeled using the juxtacellu-
lar technique, the cholinergic neurons discharge maxi-
mally in association with cortical activation during active
waking and rapid eye movement (REM) sleep [3,4]. They
cease firing in association with cortical slow wave activity
during quiet, non-REM (NREM) sleep. The cholinergic
cells also express c-Fos following continuous waking
imposed by sleep deprivation (SD), whereas they do not
express c-Fos following sleep in control (SC) or recovery
Published: 22 February 2007
BMC Neuroscience 2007, 8:15 doi:10.1186/1471-2202-8-15
Received: 15 August 2006
Accepted: 22 February 2007
This article is available from: http://www.biomedcentral.com/1471-2202/8/15
© 2007 Modirrousta et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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(SR) conditions, comprised of >75% sleep of which ~90%
is quiet, NREM sleep [5]. Their inactivity during NREM
sleep could be imposed through inhibition by GABA [6,7]
that can be released from co-distributed BF GABAergic
neurons which discharge in association with slow wave
activity [8-10] and express c-Fos in association with sleep
[5].
As evidenced by increases in the amount of sleep and in
the power of slow wave activity that occur following dep-
rivation, sleep is considered to be under homeostatic con-
trol [11-13]. Such control could be determined by similar
processes that serve to maintain long term stability in the
excitability and activity of neurons and their circuits
[14,15]. According to this homeostatic process, prolonged
activity results in decreased excitability, whereas pro-
longed inactivity results in increased excitability.
Although these changes are mediated by plastic changes
in excitatory transmission [16,17], they are also impor-
tantly mediated by reciprocal, plastic changes in inhibi-
tory transmission [15]. In cultured neurons, increased
activity (stimulated by blocking K+ channels or GABA
A
Rs)
results in increases in the density of GABA
A
Rs [18]. Con-
versely, abolition of activity (by blocking sodium chan-
nels or glutamatergic receptors) results in decreases in
density of GABA
A
Rs and parallel decreases in amplitude of
miniature inhibitory postsynaptic currents (mIPSCs)
[18,19]. We thus envisaged that the changes in activity
that occur in specific cell groups during waking and sleep
could be similarly associated with dynamic changes in
GABA
A
Rs and resulting inhibition.
With the knowledge that BF cholinergic neurons are active
and express c-Fos during continuous waking with sleep
deprivation (SD) and are inactive and do not express c-Fos
during sleep with sleep control or recovery (SC or SR)
(above, [5]), we investigated whether these changes in
activity might be associated with changes in GABA
A
Rs. We
examined immunohistochemical staining for the β
2–3
subunits of GABA
A
Rs because immunostaining for the β
2–
3
subunits was previously shown to be present on rat
cholinergic basal forebrain neurons [20] and to be altered
in density or distribution on cortical neurons as a function
of activity in previous in vitro and in vivo studies
[19,21,22]. Moreover, mRNA for the β
3
subunit GABA
A
R
in hypothalamus was also reported to change in hypotha-
lamus as a result of sleep deprivation in a preliminary
study [23]. Across conditions of SC, SD and SR, we first
examined in triple-immunostained material whether c-
Fos expressing and non-expressing, choline acetyltrans-
ferase (ChAT)-immunopositive (+) cells in the magnocel-
lular preoptic nucleus (MCPO) were immunostained for
β
2–3
GABA
A
Rs. We subsequently employed dual-immu-
nostained material to measure the luminance of immun-
ofluorescent staining for β
2–3
GABA
A
Rs on ChAT+ cells
across conditions.
Results
Sleep in SC, SD and SR conditions
As in our previous experiments [5], rats in the sleep con-
trol group (SC), which had undisturbed sleep or waking
for 3 hours before sacrifice (at 1500 h), slept the majority
of time (75.75 ± 0.61 %, mean ± S.E.M.); rats in the
deprived group (SD) did not sleep (0 %) and remained
quietly awake; and rats in the SR group that were allowed
to recover sleep in the afternoon after 3 hours deprivation
in the morning, slept >90% (92.87 ± 1.92 %) of the time
prior to sacrifice (with a significant main effect of condi-
tion according to nonparametric, Kruskal-Wallis test sta-
tistic = 8.22, df = 2, p < 0.05). The major proportion of
time for the SC and SR groups was spent in NREM sleep
(68.68 ± 0.10% in SC and 80.13 ± 0.58% in SR) and a
minor proportion in REM sleep (7.10 ± 0.66% in SC and
12.73 ± 1.40% in SR of total time).
c-Fos and GABA
A
R immunostaining in cholinergic cells
across conditions
Within sections triple-immunostained for c-Fos, ChAT
and β
2–3
GABA
A
Rs, ChAT immunopositive (+) cells which
expressed c-Fos were present in SD brains within the
MCPO in small numbers and virtually absent in SC and
SR brains, as previously reported for all BF cholinergic
nuclei [5]. In the SD brains, c-Fos+/ChAT+ cells appeared
positively immunostained for the β
2–3
GABA
A
R, which
was concentrated over the plasma membrane of the cell
(Fig. 1A). In c-Fos-negative/ChAT+ cells in the SD and
other groups, the GABA
A
R labeling was variable and par-
ticularly in the SC and SR groups not always visible, and
thus presumably below the threshold for immunohisto-
chemical detection. Using stereological random sampling
and judging whether the sampled ChAT+ cells appeared
either immuno-positive or -negative for β
2–3
GABA
A
R
immunostaining irrespective of c-Fos staining across
groups, the double-labeled ChAT+/GABA
A
R+ cells were
found to be more prevalent in the SD brains than in SC
and SR brains (Fig. 2). From stereological samples and
counts, the proportions of ChAT+ cells which were judged
to be GABA
A
R+ differed significantly across groups (F =
7.49, df = 2, df
error
= 7, p = 0.02) and were significantly
higher in the SD group (52.0 ± 0.03%) than in the SC
group (20.9 ± 0.05%; according to post hoc tests with
Tukey corrections for multiple comparisons, p 0.05).
The proportions in the SR group (31.8 ± 0.09%) were
intermediate between the SC and SD groups. This differ-
ence in receptor labeling was then assessed quantitatively
in material dual-immunostained for ChAT and the β
2–3
GABA
A
R in order to maximize the GABA
A
R staining,
which was partially attenuated by the triple-immunos-
taining procedure used for c-Fos.
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C-Fos expression and GABA
A
R labeling in BF cholinergic cells across conditionsFigure 1
C-Fos expression and GABA
A
R labeling in BF cholinergic cells across conditions. A. From triple-immunostained sections, a BF
cell triple-labeled for c-Fos (with DAB-Ni in gray, A1), ChAT (with Cy2 in green) and β
2–3
GABA
A
R (with Cy3 in red, A2)
from an SD brain. Note prominent GABA
A
R labeling over the plasma membrane of the cell. B. From dual-immunostained sec-
tions, a BF cell double-labeled for ChAT (with Cy2, B1) and β
2–3
GABA
A
R (with Cy3, B2) from an SD brain and showing the
prominent labeling along the membrane of the soma and proximal dendrites along with the boxes (in yellow) that were used
for luminance measurements of the GABA
A
R labeling over the plasma membrane (on two sides) and the nucleus. The lumi-
nance of the nucleus, which was considered to represent and thus control for nonspecific, background fluorescence, was sub-
tracted from the mean luminance of the membrane of each cell as a measure of intensity membrane GABA
A
R (Fig. 3). C-E. BF
cells dual-immunostained for ChAT (Cy2, C1, D1, E1) and β
2–3
GABA
A
R (Cy3, C2, D2, E2) from representative brains
(selected according to mean values of membrane intensity per condition) of SC, SD and SR groups (Fig. 3). Scale bar, 20 µm.
Abbreviations: SC, sleep control; SD, sleep deprived; SR, sleep recovery.
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Intensity of GABA
A
R membrane staining over cholinergic
cells across conditions
In dual-immunostained material, the β
2–3
GABA
A
R immu-
nostaining was prominent over the membrane of the
soma and proximal dendrites of numerous ChAT+ cells,
particularly in brains from the SD group (Fig. 1B). As
described in previous in vivo studies of GABA
A
R labeling
for β
2–3
, as well as other, subunits [20,24], the fluorescent
staining was commonly distributed along the full mem-
brane of the cell body and proximal dendrites. To deter-
mine if the intensity of GABA
A
R labeling on cholinergic
cells was different across SC, SD and SR conditions, lumi-
nance measures of the GABA
A
R immunofluorescence over
the membrane were performed and corrected for nonspe-
cific background fluorescence by subtracting the lumi-
nance measured over the nucleus of each cell in the
acquired images (Fig. 1B2, see Methods). In randomly
sampled ChAT+ cells (~25 cells per brain) obtained by
stereological sampling within the MCPO (from three lev-
els/sections per brain in 10 brains), the intensity of the
membrane GABA
A
R labeling was found to differ signifi-
cantly across conditions (F = 11.44, df = 2, df
error
= 224, p
< 0.001; Fig. 3). It was significantly higher in the SD group
than in both the SC and SR groups (p 0.05; post hoc tests
with Tukey corrections for multiple comparisons).
The quantitative changes in the intensity of membrane β
2–
3
GABA
A
R immunostaining were evident in the images
(Fig. 1C–E) taken from ChAT+ cells having intensity
measures near the average values for the SC, SD and SR
groups (Fig. 3). The images illustrate how under control
conditions (SC), the GABA
A
R labeling was on average very
low and under the threshold for immunohistochemical
detection (Fig. 1C2), how under deprived conditions
(SD), it was on average very intense (Fig. 1D2), and how
under recovery conditions (SR) following deprivation, it
was also on average relatively low (Fig. 1E2).
Discussion
The present results show that GABA
A
R labeling on BF
cholinergic neurons increases during periods of waking
when the cells are active and decreases during periods of
sleep when they are inactive. They suggest that as with
neurons in culture, the cholinergic neurons in the brain
might undergo homeostatic regulation of their excitability
as a function of prior activity through changes in GABA
A
Rs
and associated inhibition across the sleep-waking cycle.
We first noted here that cholinergic cells which express c-
Fos and are thus particularly active during SD [5] show
membrane immunostaining for the β
2–3
GABA
A
R. Such
labeling was variable on cholinergic cells which did not
express c-Fos in the SD brains and often undetectable on
such cells in SC and SR brains. Across groups, GABA
A
R
labeling was judged to be positive on the majority of
cholinergic cells in the SD group, and only on the minor-
Map of BF cholinergic cells with GABA
A
R labeling across conditionsFigure 2
Map of BF cholinergic cells with GABA
A
R labeling across conditions. ChAT+ cells plotted in single sections (of triple-immunos-
tained series, Fig. 1A) through the middle level of the MCPO (~A8.2) from representative brains of SC (A), SD (B) and SR (C)
groups. Presumed to reflect a threshold for immunohistochemical detection, GABA
A
R labeling was judged to be negative (open
circles) or positive (filled circles) in each ChAT+ cell within the section. Note the ChAT+/GABA
A
R+ cells are most prevalent
in the SD brain. Abbreviations: f, fornix; LPO, lateral preoptic area; MCPO, magnocellular preoptic area; MPO, medial preoptic
area; SI, substantia innominata.
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ity in the SC and SR groups. This labeling was considered
to reflect different numbers or concentrations of GABA
A
Rs
which accordingly would or would not reach threshold
for immunohistochemical detection. We presume that the
presence of c-Fos along with GABA
A
R labeling in neurons
in the SD condition is a reflection of their prolonged activ-
ity during continuous waking. We also presume that the
lack of c-Fos expression and parallel paucity of GABA
A
R
labeling in the SC and SR conditions are commonly due
to the amount of time spent in quiet, NREM sleep (~124
and 144/180 min on average respectively), during which
the cholinergic cells would be silent [4], and not signifi-
cantly affected by the amount of time spent in active, REM
sleep (~13 and 23/180 min), during which the cholinergic
neurons would be firing [4]. We accordingly assume that
the changes in GABA
A
R labeling in the different groups
reflect different levels of activity by the cholinergic neu-
rons as predominantly high during waking in SD vs. pre-
dominantly low during sleeping in SC and SR conditions.
According to luminance measures of β
2–3
GABA
A
R immu-
nostaining across groups in dual-immunostained mate-
rial, we found that the intensity of membrane GABA
A
R
labeling was significantly greater in the SD, waking rats
than in the SC, sleeping rats, suggesting that the labeling
was a function of the preceding activity by the cholinergic
cells. These in vivo results are similar to those in culture
showing that prolonged increases in activity result in
increased density of GABA
A
Rs along with increased inhib-
itory currents on hippocampal neurons [18]. Previous in
vivo studies in the hippocampus also showed parallel
increases in GABA
A
Rs and inhibitory currents following
increased activity through experimentally induced sei-
zures [21]. Here, the presumed increased activity of the
cholinergic cells during 3 hours continuous waking
imposed by sleep deprivation during the day would be
neither as prolonged nor as extreme as that evoked in the
in vitro and in vivo models, however could represent a
more natural condition and resulting homeostatic adjust-
ment to sustained activity that could occur during the nat-
ural sleep-waking cycle or disturbances to that cycle.
We also found that the intensity of membrane β
2–3
GABA
A
R immunostaining on the cholinergic cells was sig-
nificantly decreased following recovery sleep in the SR
condition as compared to the SD condition. In view of the
virtual silence of cholinergic neurons during NREM, slow
wave sleep [4], these in vivo results are parallel to results in
culture showing decreases in GABA
A
R labeling and inhib-
itory currents following abolition of activity in cortical
neurons [18,19]. Although not statistically significantly
different, the intensity of GABA
A
R labeling in the SR con-
dition was not as low as that in the SC condition, possibly
due to a variably incomplete return to control levels dur-
ing recovery following the increase which would have
occurred with the preceding 3 hour deprivation.
In the immunofluorescent images, we found that the β
2–3
GABA
A
R immunostaining was concentrated over the
plasma membrane of the cell, often distributed along the
entire membrane of the soma and proximal dendrites and
altered in intensity over the membrane as a function of
condition. This relatively continuous, though neither
homogeneous nor clustered, pattern of in vivo staining for
the β
2–3
, as well as other, subunit GABA
A
Rs has been
described previously for in vivo staining of basal forebrain
as well as other neurons in the brain [20,24]. Such mem-
brane staining is presumed to reflect functional receptors,
which would contain β subunits [21]. The continuous dis-
tribution likely reflects the presence of many, including
the β
2–3
subunit, GABA
A
Rs in the extrasynaptic as well as
synaptic membrane of the cells [25-27]. Both extrasynap-
Intensity membrane GABA
A
R labeling on BF cholinergic cells across conditionsFigure 3
Intensity membrane GABA
A
R labeling on BF cholinergic cells
across conditions. Values (mean ± SEM per group) represent
luminance measures (brightness of image acquired through
camera) of GABA
A
R immunofluorescence (Cy3) over the
plasma membrane (average two sides) minus that over the
nucleus of each ChAT+ cell sampled (see Fig. 1B). The inten-
sity of membrane GABA
A
R labeling was significantly higher in
the SD group than in the SC (*) and SR (†) groups (p 0.05,
based upon Tukey adjusted post-hoc comparisons, see text).
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tic and synaptic membrane receptors nonetheless reflect
functional GABA
A
Rs, which respectively mediate tonic
and phasic currents [28]. Accordingly, the changes in
intensity of the β
2–3
GABA
A
R immunostaining over the
membrane observed in the present study could reflect
changes in functional receptors. Increases in membrane
GABA
A
Rs would be associated with marked enhancement
of inhibition since the GABA
A
Rs in the central nervous
system are believed to be commonly fully saturated by the
release of GABA from one synaptic vesicle [29]. Indeed,
the number of surface β
2–3
subunit GABA
A
Rs has been
shown to be directly correlated with the amplitude of
inhibitory currents [21,22]. Although it cannot be sur-
mised how the changes in membrane GABA
A
Rs occurred
in the present study, it is known that rapid increases in cell
surface GABA
A
Rs can occur by their recruitment to the
membrane from intracellular stores [21,22]. Reciprocally,
rapid decreases can occur by internalization or endocyto-
sis of the receptors [30,31]. Given preliminary reports of
increases in mRNA for β
3
GABA
A
Rs following 7 hours of
sleep deprivation and varying levels of the mRNA in rela-
tion to the sleep cycle [23], it is also possible that the
changes in membrane GABA
A
Rs involve changes in pro-
tein synthesis. Indeed, increases or decreases in mem-
brane GABA
A
Rs can occur through multiple mechanisms
involving differential intracellular trafficking along with
changes in protein synthesis, phosphorylation and degra-
dation [30,31].
Conclusion
According to our results and interpretations, membrane
GABA
A
Rs on the BF cholinergic neurons would progres-
sively increase as a function of activity during waking and
progressively decrease as a function of inactivity during
NREM, slow wave sleep. Since the cholinergic neurons
stimulate fast gamma activity and attenuate slow delta
activity on the cortex [1,32,33], an increase in GABA-
mediated inhibition of the cholinergic cells following
waking would be associated with decreases in fast and
increases in slow cortical activity, and a decrease in GABA-
mediated inhibition during sleep would be associated
with reciprocal increases in fast and decreases in slow cor-
tical activity. Such hypothetical changes parallel those
measured in slow, delta activity which varies as a function
of prior waking, being maximal at the onset of sleep and
decreasing progressively during sleep [12]. The dynamic
changes in GABA
A
Rs on cholinergic BF neurons would
thus reflect as well as participate in the homeostatic regu-
lation of cerebral activity across the sleep-waking cycle.
Methods
All procedures were approved by the McGill University
Animal Care Committee and conformed to the Canadian
Council on Animal Care.
Sleep deprivation
Male Wistar rats (average ~240 g at termination of experi-
ment, corresponding to ~50 days old) were housed indi-
vidually with free access to food and water at all times
with a 12:12 light/dark schedule (lights on from 700 to
1900 h). As previously described [5], the rats were
deprived of sleep and observed in their home cages so as
to avoid stress. All rats were sacrificed at 1500 h and were
previously submitted respectively to 1) total sleep depri-
vation (SD) for 3 hours (1200 to 1500 h, n = 3), 2) total
sleep deprivation for 3 hours (900 to 1200 h) followed by
sleep recovery (SR) for 3 hours (1200 to 1500 h, n = 3), or
3) undisturbed sleep and waking as sleep control (SC) for
3 hours (1200 to 1500 h, n = 4). Rats in the SD and SR
groups were deprived of sleep by being gently touched
with a paint brush upon closure of their eyes. Sleep-wake
states were scored every 20 sec by behavioral observations
as wake, NREM sleep or REM sleep. Behavioral scoring
was used in order to avoid the stress of surgery and tether-
ing for recording and has been found to be sufficient for
scoring the major states of wake, NREM (behaviorally
quiet) sleep and REM (behaviorally active with twitches)
sleep in normal rats [5,34,35]. At the end of the experi-
ment (1500 h), rats were immediately killed under pento-
barbital anesthesia (100 mg/kg, i.p.) by intra-aortic
perfusion with a fixative solution of 3% paraformalde-
hyde.
Immunohistochemistry
Following immersion in a 30% sucrose solution, brains
were frozen and stored at -80°C. They were cut in coronal
sections at 20 µm thickness and collected at 800 µm inter-
vals as multiple series through the basal forebrain. One
series of sections from each brain (n = 10) was immedi-
ately processed for triple immunohistochemical staining
of c-Fos, ChAT and GABA
A
R. The remaining series were
frozen in 30% glycerol-ethylene glycol solution and
stored at -20°C. Following stereological analysis (below)
of the triple-immunostained series which revealed
changes in GABA
A
R labeling of ChAT+ cells, one series of
sections from all brains (n = 10) were simultaneously
processed for dual immunofluorescent staining for ChAT
and GABA
A
R in order to maximize the receptor labeling
and allow for quantitative measures of the labeling using
luminance measures (below).
Antibodies were employed for triple or dual immunohis-
tochemical staining for c-Fos, ChAT and GABA
A
R. For c-
Fos, a rabbit (Rb) antiserum (1:10,000, Ab-5, PC38,
Oncogene Research Products, San Diego, CA) was
employed and revealed using the peroxidase-anti-peroxi-
dase (PAP) technique (Rb PAP and donkey (Dky) anti-Rb
from Jackson ImmunoResearch Laboratories, West Grove,
PA) processed with diaminobenzidine-nickel (DAB-Ni).
For ChAT, a Rb antiserum (1:1000, AB143, Chemicon
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International, Temecula, CA) was employed and revealed
by immunofluorescent staining with Dky Cy2-conjugated
anti-Rb antiserum (Jackson). For the GABA
A
R, a mouse
(Ms) monoclonal antibody against the β
2–3
chain subu-
nits (1:100, MAB341, Chemicon) was employed and
revealed using immunofluorescent staining with Dky
Cy3-conjugated anti-Ms antiserum (Jackson). For the tri-
ple-immunostained series, the sections were first incu-
bated with the c-Fos antibody overnight at room
temperature and processed by PAP staining with DAB-Ni,
which produced black staining over the nucleus. For both
triple- and dual-immunostained series, the sections were
co-incubated with ChAT and GABA
A
R antibodies for three
nights at 4°C followed by co-incubation with Cy2- and
Cy3-conjugated secondary antisera for 2 hours. The ChAT
Cy2 staining was seen within the cytoplasm, and the
GABA
A
R Cy3 staining over the plasma membrane (see
Results).
Microscopic analysis
Sections were viewed by light and fluorescence micros-
copy with a Nikon Eclipse E800 microscope equipped
with an x/y/z movement-sensitive stage and digital cam-
era (Optronics, Microfire S99808, Goleta, CA) attached to
a computer. Fluorescence was viewed and acquired using
appropriate filter sets for Cy2 (bandpass excitation filter,
460–500 nm; longpass dichromatic mirror, 505 nm;
bandpass emission filter, 510–560 nm) and Cy3 (band-
pass excitation filter, 510–560 nm; longpass dichromatic
mirror, 565 nm; longpass emission filter, 590 nm). Cell
counts and image acquisition were performed by system-
atic random sampling using StereoInvestigator (Micro-
BrightField, MBF, Williston, VT). On the acquired images,
measurements were performed for brightness of the fluo-
rescence, thus called luminance since it refers to the
brightness of the light transmitted through the camera,
using Neurolucida software (MBF). For these applica-
tions, a computer resident atlas was employed through
the rat BF (~Anterior, A7.0 to A11.0 from interaural zero)
[5].
Cell counts
In triple-immunostained series, ChAT+ cells were exam-
ined for being positively labeled for the β
2–3
GABA
A
R and
for c-Fos (Fig. 1A). Single-, double- and triple-labeled cells
were plotted and counted in all brains (n = 10) through
the MCPO at three levels (corresponding to three sections
from the rostral to caudal extent of the nucleus at ~A9.0,
A8.2 and A7.4) using the Optical Fractionator program of
StereoInvestigator to provide unbiased sampling and
resulting estimates of proportions of labeled cells through
the nucleus. Within the stereology program, cells were
counted under a 60× oil objective (with 1.4 numerical
aperture) using a counting frame of 125 × 125 µm, which
yielded 3 or more cells counted per frame, and sampling
grid of 250 × 250 µm, which yielded 10 counting sites or
more per section and >35 counting sites for the MCPO per
brain. In one representative brain from each series, cells
were mapped and counted using a sampling grid of 125 ×
125 µm for representation of all cells per section. Cells
that came into focus beneath the surface of each section
were counted within a counting block of 8 µm in depth
(in the mounted and dehydrated sections that were on
average 10 µm thick).
Luminance measurements
In dual-immunostained series, images of ChAT+ cells and
their β
2–3
GABA
A
R immunofluorescence were acquired
using the 8-bit setting of the digital camera, which thus
provides a gray scale of 0–256 levels for luminance meas-
ures in Neurolucida. For all images, the fluorescence illu-
mination was the same, and the settings of the camera
were the same (gain of 4 and 100 ms exposure). Image
acquisition was made as rapidly as possible for each cell
so as to avoid bleaching of the fluorescence. In each brain
(n = 10), random sampling through three levels (corre-
sponding to three sections at ~A9.0, A8.2 and A7.4) of the
MCPO was performed using StereoInvestigator. Within
the Optical Fractionator program, a counting frame of 50
× 50 µm and grid of 250 × 250 µm were employed for
sampling ~25 sites and thus cells per brain. In each site,
the ChAT+ cell which was the closest to the centre of the
counting frame was selected for image acquisition. The
images of the ChAT+ cell and its GABA
A
R labeling were
acquired using a 60× oil objective (Fig. 1B–E). Luminance
measurements were performed on the acquired fluores-
cent images using Neurolucida, which provides data in
arbitrary units (on a gray scale of 0 – 256). For this appli-
cation, a box was created with a length of 5 µm and a
width of 1 µm, which was established as the maximal
length and thickness of GABA
A
R staining that could be
consistently measured over the plasma membrane of the
ChAT+ cells. In each image, boxes were positioned paral-
lel to the long axis of the cell soma over the plasma mem-
brane (on two sides) and over the nucleus (Fig. 1B2) for
collection of average luminance values within each box.
The average luminance values (mean ± SEM for measure-
ments over 227 cells in 10 brains) were 49.18 ± 0.88 for
the plasma membrane and 45.10 ± 0.81 for the nucleus.
For each cell, the intensity of membrane GABA
A
R immu-
nostaining was calculated by taking the average mem-
brane luminance (of the two sides) and subtracting the
luminance of the nucleus, which was considered to repre-
sent nonspecific immunofluorescence and thus by sub-
traction to control for variations in background staining
across cells, sections and brains.
Statistical analysis and presentation
Data were analyzed across conditions by the nonparamet-
ric Kruskal-Wallis test for sleep-wake values, which
BMC Neuroscience 2007, 8:15 http://www.biomedcentral.com/1471-2202/8/15
Page 8 of 9
(page number not for citation purposes)
included zero values, or by one way ANOVA followed by
Tukey corrected post-hoc comparisons for cell counts and
luminance measures (Systat, v10.2, Richmond, CA). Fig-
ures were composed using Adobe Photoshop and Illustra-
tor Creative Suite (Adobe, San Jose, CA). Images are
presented as they were acquired using the 8-bit setting of
the camera which is compatible with the 8-bit image
option of Adobe Photoshop and without applying any
adjustment for brightness or contrast.
Abbreviations
BF, basal forebrain
ChAT, choline acetyltransferase
GABA
A
R, GABA
A
receptor
MCPO, magnocellular preoptic area
mIPSC, miniature inhibitory postsynaptic current
NREM, nonREM
REM, rapid eye movement
SC, sleep control
SD, sleep deprived
SR, sleep recovery
Authors' contributions
MM conducted all the experiments, analyzed the data and
drafted the manuscript; LM provided technical assistance;
BEJ directed the experiments, reviewed all data analysis
and wrote the final manuscript. All authors read and
approved the manuscript.
Acknowledgements
Supported by Canadian Institutes of Health Research (CIHR 13458) and
U.S. National Institutes of Health (NIH RO1 MH-60119-01A).
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    • "The secondary detrimental manifestations associated with sleep deprivation include anxiety like behavior, cognitive deficits, oxidative stress (via accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, neuroinflammation etc. that have all been individually responsible for affecting the GABAergic transmission. Not only this, the various allosteric binding sites of GABAA receptor have also served as potential therapeutic targets for the management of SD induces anxiety like problems (Modirrousta et al., 2007; Tadavarty et al., 2011). In Abbreviations: BF, Basal forebrain; CRP, C-Reactive Protein; DNA, deoxyribonucleic acid; GABA, gamma amino butyric acid; NO, nitric oxide; GSH, reduced glutathione; H 2 O 2 , hydrogen peroxide; MDA, malondialdehyde; SD, Sleep Deprivation, TNF, tumor necrosis factor; VLPO, Venterolateral preoptic. "
    [Show abstract] [Hide abstract] ABSTRACT: Rationale- Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective- The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-hours sleep deprivation induced anxiety like behaviour, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods- Male laca mice were sleep deprived for 72-hours by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100 and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl- channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting five days prior to 72-hours sleep deprivation period. Various behavioural (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumour Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results- 72-hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behaviour, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioural, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl- channel inhibitor as well as GABA-benzodiazepine receptor inhibitor, significantly reversed the protective effect of P. quinquefolius (100 mg/kg) in 72-hours sleep deprived animals (P<0.05). However, pretreatment with GABAA agonist, potentiated Panax quinquefolius’s protective effect which was significant as compared to their effect per se (p<0.05). Conclusion- GABA-ergic mechanism could be involved in the neuroprotective effect of P. quinquefolius against sleep deprivation induced anxiety-like behaviour, oxidative stress, mitochondrial dysfunction, HPA axis activation and neuroinflammation.
    Full-text · Article · Feb 2016
    • "However, endogenous GABA release in the BF is high during NREM sleep compared to waking and REM sleep (Nitz and Siegel 1996; Vanini et al. 2012). Infusion of GABA agonists into the BF promotes sleep (Manfridi et al. 2001), infusion of GABA A receptor antagonists increases ACh release (Vazquez and Baghdoyan 2003), and SD increases GABA receptor expression on ACh neurons (Modirrousta et al. 2007), suggesting that GABA released in the BF has a net sleep-promoting influence. Consequently, the ALM-induced GABA release that we observed during Waking and Mixed states likely comprises an important part of ALM's ability to induce sleep, and does so in a way that mimics the neurochemical events normally associated with the transition to sleep. "
    [Show abstract] [Hide abstract] ABSTRACT: Hypocretin/orexin (HCRT) neurons provide excitatory input to wake-promoting brain regions including the basal forebrain (BF). The dual HCRT receptor antagonist almorexant (ALM) decreases waking and increases sleep. We hypothesized that HCRT antagonists induce sleep, in part, through disfacilitation of BF neurons; consequently, ALM should have reduced efficacy in BF-lesioned (BFx) animals. To test this hypothesis, rats were given bilateral IgG-192-saporin injections, which predominantly targets cholinergic BF neurons. BFx and intact rats were then given oral ALM, the benzodiazepine agonist zolpidem (ZOL) or vehicle (VEH) at lights-out. ALM was less effective than ZOL at inducing sleep in BFx rats compared to controls. BF adenosine (ADO), γ-amino-butyric acid (GABA), and glutamate levels were then determined via microdialysis from intact, freely behaving rats following oral ALM, ZOL or VEH. ALM increased BF ADO and GABA levels during waking and mixed vigilance states, and preserved sleep-associated increases in GABA under low and high sleep pressure conditions. ALM infusion into the BF also enhanced cortical ADO release, demonstrating that HCRT input is critical for ADO signaling in the BF. In contrast, oral ZOL and BF-infused ZOL had no effect on ADO levels in either BF or cortex. ALM increased BF ADO (an endogenous sleep-promoting substance) and GABA (which is increased during normal sleep), and required an intact BF for maximal efficacy, whereas ZOL blocked sleep-associated BF GABA release, and required no functional contribution from the BF to induce sleep. ALM thus induces sleep by facilitating the neural mechanisms underlying the normal transition to sleep.
    Full-text · Article · Nov 2014
    • "Collectively, these results demonstrate the importance of GABA A Rs, and the b3 subunit in particular, for the regulation of both SWS and REMS sleep, but they do not provide information about the anatomic and cellular location of the relevant GABA A Rs. In addition to our data pointing to the PF region of the posterior hypothalamus, an increased immunostaining for the b2/3 subunits of GABA A R was found following SD on neuronal membranes of basal forebrain cholinergic neurons [64]. Thus, the regulation of b3 subunit levels in multiple brain regions may contribute to the regulation of sleep. "
    [Show abstract] [Hide abstract] ABSTRACT: Sleep-wake behavior is regulated by a circadian rhythm, homeostatically and by additional mechanisms that determine the timing of slow-wave sleep and rapid eye movement sleep (REMS) episodes. The posterior hypothalamus coordinates the neural and humoral signals with the rest-activity cycle. It contains wake-active neurons, and is a site where stimulation of inhibitory GABAA receptors promotes sleep, whereas their antagonism enhances wakefulness. We explored whether GABAergic mechanisms present in the posterior hypothalamus contribute to the homeostatic and other aspects of sleep-wake regulation. Using micropunches of tissue extracted from either the perifornical (PF) or dorsomedial (DM) regions of the posterior hypothalamus of rats, we determined that mRNA levels for selected subunits of GABAA receptors (β1, β3 and ε) were higher at the end of the active period or following sleep deprivation, when the need for sleep is high, than after several hours of sleep, when sleep need is partially fulfilled. Such a pattern was present in the PF region only, and was consistent with changes in β1 subunit and GABA synthesizing enzyme (GAD) protein levels. In contrast, in the DM region, the levels of GABAA receptor subunit mRNAs and proteins (α1, α2, β1) and GAD varied with circadian time, but were not responsive to sleep deprivation. Separate experiments with sleep-wake monitoring and local perfusion of the PF region with the GABAA receptor antagonist bicuculline revealed that the antagonist had a weaker sleep-reducing effect when sleep need was enhanced by sleep deprivation and that the increased amount of REMS characteristic of the late sleep period was dependent on endogenous GABAergic inhibition. These results support the concept that a varying magnitude of GABAergic inhibition exerted within the PF region contributes to the homeostatic regulation of sleep and shapes its temporal pattern, whereas GABAergic mechanisms in the DM region contribute to circadian regulation.
    Full-text · Article · Jan 2014
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