Noradrenaline acting on α1-adrenoceptor mediates REM sleep deprivation-induced increased membrane potential in rat brain synaptosomes

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Neurochemistry International (Impact Factor: 3.09). 03/2008; 52(4-5):734-40. DOI: 10.1016/j.neuint.2007.09.002
Source: PubMed
ABSTRACT
We hypothesized that one of the functions of REM sleep is to maintain brain excitability and therefore, REM sleep deprivation is likely to modulate neuronal transmembrane potential; however, so far there was no direct evidence to support the claim. In this study a cationic dye, 3,3'-diethylthiacarbocyanine iodide was used to estimate the potential in synaptosomal samples prepared from control and REM sleep deprived rat brains. The activity of Na-K-ATPase that maintains the transmembrane potential was also estimated in the same sample. Further, the roles of noradrenaline and alpha1-adrenoceptor in mediating the responses were studied both in vivo as well as in vitro. Rats were REM sleep deprived for 4 days by the classical flower-pot method; large platform and recovery controls were carried out in addition to free-moving control. The fluorescence intensity increased in samples prepared from REM sleep deprived rat brain as compared to control, which reflected synaptosomal depolarization after deprivation. The Na-K-ATPase activity also increased in the same deprived sample. Furthermore, both the effects were mediated by noradrenaline acting on alpha1-adrenoceptors in the brain. This is the first direct evidence showing that REM sleep deprivation indeed increased neuronal depolarization, which is the likely cause for increased brain excitability, thus supporting our hypothesis and the effect was mediated by noradrenaline acting through the alpha1-adrenoceptor.

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Available from: Gitanjali Das, Aug 20, 2015
Noradrenaline acting on a1-adrenoceptor mediates REM sleep
deprivation-induced increased membrane potential
in rat brain synaptosomes
Gitanjali Das, Birendra Nath Mallick
*
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Received 22 June 2007; received in revised form 1 September 2007; accepted 5 September 2007
Available online 11 September 2007
Abstract
We hypothesized that one of the functions of REM sleep is to maintain brain excitability and therefore, REM sleep deprivation is likely to
modulate neuronal transmembrane potential; however, so far there was no direct evidence to support the claim. In this study a cationic dye, 3,3
0
-
diethylthiacarbocyanine iodide was used to estimate the potential in synaptosomal samples prepared from control and REM sleep deprived rat
brains. The activity of Na–K–ATPase that maintains the transmembrane potential was also estimated in the same sample. Further, the roles of
noradrenaline and a1-adrenoceptor in mediating the responses were studied both in vivo as well as in vitro. Rats were REM sleep deprived for 4
days by the classical flower-pot method; large platform and recovery controls were carried out in addition to free-moving control. The fluorescence
intensity increased in samples prepared from REM sleep deprived rat brain as compared to control, which reflected synaptosomal depolarization
after deprivation. The Na–K–ATPase activity also increased in the same deprived sample. Furthermore, both the effects were mediated by
noradrenaline acting on a1-adrenoceptors in the brain. This is the first direct evidence showing that REM sleep deprivation indeed increased
neuronal depolarization, which is the likely cause for increased brain excitability, thus supporting our hypothesis and the effect was mediated by
noradrenaline acting through the a1-adrenoceptor.
# 2007 Elsevier Ltd. All rights reserved.
Keywords: DiSC
2
; Excitability; Membrane depolarization; Na–K–ATPase; Noradrenaline; REM sleep deprivation; Synaptosomes
Rapid eye movement (REM) sleep plays a crucial role in the
development and sustenance of the central nervous system as
evidenced by its presence across evolution at least from birds to
mammals (Amlaner and Ball, 1994; Zepelin, 1994; Siegel et al.,
1999) and its decrease with age (Gaudreau et al., 2005). REM
sleep is reported to decrease in several diseases including
Alzheimer’s, Parkinson’s (Montplaisir et al., 1995; Gagnon
et al., 2002) and in several psychiatric disorders (Vogel, 1999).
Its loss has been associated with several signs and symptoms
like increased anxiety, aggression, irritability, confusion, loss of
concentration (for review see Gulyani et al., 2000) and
increased sensitiveness to tactile stimuli where subjects tend to
flinch, jump and squeal (Kushida et al., 1989). REM sleep loss
has been reported to affect mood; behavior and threshold for
electroconvulsive shock in both animal and human subjects
(Kushida et al., 1989; Gulyani et al., 2000; Clark, 2005). Recent
studies have shown that longer duration of REM sleep
deprivation (REMSD) results in morphological changes in
neurons (Majumdar and Mallick, 2005) and neuronal death
(Biswas et al., 2006; Cordova et al., 2006). Based on these
altered behavioral and physiological changes we hypothesized
that REMSD alters brain excitability and as a corollary we
proposed that one of the functions of REM sleep is to maintain
the threshold of neuronal and brain excitability as well as
responsiveness (Mallick et al., 1994); however, its mechanism
of action was unknown.
A reciprocal relationship exists between the level of
excitability and membrane potential in neurons, where the
latter is the cause and an estimate of the former. The higher the
positivity of the intracellular potential, greater is the excitability
level and lower is the threshold of responsiveness of the neuron.
As a possible mechanism of action for such changes in
excitability we showed that REMSD increases (Gulyani and
Mallick, 1993) the activity of the neuronal membrane bound
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* Corresponding author. Tel.: +91 11 26704522; fax: +91 11 26717558.
E-mail address: remsbnm@yahoo.com (B.N. Mallick).
0197-0186/$ see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2007.09.002
Page 1
enzyme Na–K–ATPase, a key factor that maintains transmem-
brane potential. On the other hand, REMSD increases the level
of noradrenaline (NA) in the brain (for review see Pal et al.,
2005). However, no direct study was available to show the
effect of REMSD on membrane potential of neurons, which
might be responsible for the REMSD-induced altered excit-
ability and its relationship with the increased level of NA.
Technical limitation of recording transmembrane potential of
the same neuron in vivo in freely behaving animals, before and
after a reasonable period of REMSD, is a major hurdle for such
study. Hence, in this in vivo and in vitro studies we estimated the
membrane potential using a cationic dye and correlated it with
Na–K–ATPase activity in the same synaptosomal fraction from
the control and REMSD rat brains. Furthermore, we correlated
the effect of NA in the presence and absence of its antagonist on
those parameters.
1. Methodology
1.1. Materials
Noradrenaline (NA); a 1-adrenoceptor antagonist, prazosin
(PRZ); b-adrenoceptor antagonist, propranolol (PRN); a2-
adrenoceptor agonist, clonidine (CLN); Na–K–ATPase inhi-
bitor, ouabain; mono-cationic fluorescent probe, 3,3-
diethylthiacarbocyanine iodide (DiSC
2
) and dimethyl sulfoxide
(DMSO) were procured from Sigma–Aldrich, USA. All other
chemicals were of analytical grade.
2. Animals
All the animal experimental protocols were approved by the
Institutional Animal Ethics Committee. Male inbred Wistar rats
(250–280 g), supplied with food and water ad libitum,
maintained at 12/12 h light/dark cycle were used in this study.
Standard flower-pot method was used for 4 days REMSD as
reported earlier (Gulyani and Mallick, 1993). In brief, the rats
were maintained on small (6.5 cm) platform raised over
surrounding water. To rule out non-specific effects control rats
were maintained under identical conditions for the same period
in the same room on raised larger (13 cm) platform (LPC)
surrounded by water. Free-moving home cage rats were used as
normal control (FMC) for the baseline value. Another control
set included rats deprived of REM sleep for 4 days that were
then allowed to recover from REM sleep loss for 3 days in their
normal home cages, the recovery control (REC). Thus, in each
set there was one rat each of FMC, LPC, REMSD and REC and
five such sets of studies were carried out. Additionally, in
separate series of experiments intraperitoneal injection of PRZ
and CLN were done in five different sets, each having one FMC
and one REMSD rats. For in vitro experiments synaptosomes
prepared from the FMC rat brain were treated with NA alone or
in the presence of either PRZ or PRN. Since PRN did not
prevent the NA-induced effects in in vitro studies, it was not
used for the in vivo studies. Thus, every effort was made to
minimize the use of number of rats and finally, data from 45 rats
are presented in the study.
2.1. Synaptosome preparation
The deprived as well as the control rats were decapitated
after cervical dislocation. The brains were quickly removed and
homogenized in 10 volumes of ice-cold buffer containing
0.32 M sucrose and 12 mM Tris at pH 7.4 for synaptosome
preparation from the whole brain (Mallick and Adya, 1999). In
brief, the brain homogenate was centrifuged for 5 min at
3000 g and the supernatant was further centrifuged at
11,000 g for 20 min. The pellet obtained was suspended in
1 ml of the homogenizing buffer, loaded onto a preformed
sucrose density gradient of 1.2 M and 0.8 M and ultracen-
trifuged in a swing-out rotor at 100,000 g for 2 h. The band
obtained at the interface of 1.2 M and 0.8 M sucrose was taken
as synaptosome, which was divided into two parts. One part
was re-suspended in the homogenizing buffer while the other
part was re-suspended in the HEPES buffer containing 140 mM
NaCl, 5 mM KCl, 5 mM NaHCO
3
, 1 mM MgCl
2
6H
2
O,
1.2 mM Na
2
HPO
4
, 10 mM glucose, 20 mM HEPES at pH
7.4. The former was used for assaying Na–K–ATPase activity
while the latter for synaptosomal membrane potential study.
The protein concentration in the synaptosomes was estimated
by the Lowry’s method (1951) using bovine serum albumin as
standard.
2.2. Modulation of membrane potential and Na–K–ATPase
activity by NA
The studies were conducted both in vivo and in vitro
conditions. Both membrane potential as well as Na–K–ATPase
activities were estimated from the same synaptosomal fraction
of FMC, LPC, REMSD and REC rat brains. For the in vivo
experiments either PRZ (4 mg/kg) or CLN (0.1 mg/kg) was
injected (i.p.) into both the FMC and REM sleep deprived rats
8 h before sacrifice. For the in vitro studies synaptosomes
prepared from the FMC rat whole brain were incubated with
NA (100 mM) alone or in the presence of either PRZ (50 mM)
or PRN (50 mM) and membrane potential as well as Na–K–
ATPase activities estimated.
2.3. Estimation of membrane potential
DiSC
2
is a membrane permeable mono-cationic fluorescent
dye, which accumulates within the synaptosomes that
resembles the inside of a neuron and therefore is relatively
negative (compared to the extracellular compartment). It has
been convincingly shown that DiSC
2
movement inside the cells
is directly proportional to the intracellular negativity (Wang
et al., 2001). The anions inside the synaptosomes bind to DiSC
2
and consequently quench its fluorescence. Hence, greater the
intracellular negativity more was the DiSC
2
quenching,
resulting in reduced net fluorescence intensity. Thus, intensity
of fluorescence was inversely proportional to the intracellular
anion concentration and as a corollary increased DiSC
2
-
fluorescence intensity meant membrane depolarization (Wagg-
oner, 1976; Hare and Atchison, 1992). Therefore synaptosomal
(reflection of intracellular) membrane potential was estimated
G. Das, B.N. Mallick / Neurochemistry International 52 (2008) 734–740 735
Page 2
by measuring the intensity of the cationic fluorescent probe
DiSC
2
as described previously by Perkinton and Sihra (1998)
and was expressed as fluorescence units/50 mg synaptosomes.
In brief, the DiSC
2
stock solution was made in DMSO and
working solution was prepared fresh every time by diluting the
stock solution in the HEPES buffer such that the final
concentration of the dye loaded in the synaptosomes was of
4 mM. The control and REMSD synaptosomes (50 mg) were
separately but simultaneously incubated with DiSC
2
(4 mM) for
10 min at 37 8C, following which the reaction was terminated
by addition of ice-cold HEPES buffer and the volume was made
up to 2 ml. The DiSC
2
-loaded synaptosomes were stored on ice
till the fluorescence intensity was measured using a Cary
Eclipse Spectrofluorimeter (Varian, Palo Alto, USA) with the
filters set at 646 nm and 674 nm for excitation and emission,
respectively.
2.4. Estimation of Na–K–ATPase activity
The Na–K–ATPase activity was estimated as reported earlier
(Gulyani and Mallick, 1993). Synaptosomes (40 mg) were
incubated with the reaction buffer containing 100 mM NaCl,
20 mM KCl, 5 mM MgCl
2
, 3 mM ATP and 50 mM Tris at pH
7.4 in the presence and absence of 1 mM ouabain (blocker of
Na–K–ATPase) at 37 8C for 15 min. After 15 min, the reaction
was stopped by addition of 1 ml of 10% ice-cold trichloroacetic
acid. ATP was used as the substrate and the concentration of the
liberated inorganic phosphate was estimated by the Fiske and
SubbaRow (1925) method. Thus, the ouabain sensitive ATPase
activity was estimated and expressed as mM Pi released/
mg protein h.
3. Statistics
Data has been presented as mean S.E.M. Statistical
analysis was carried out using SigmaStat 3.5 software (Jandel
Scientific, San Rafael, CA, USA). Differences between the
mean values from each experimental and treated group
compared with FMC and Ctl groups were evaluated using
analysis of variance (ANOVA-one way) coupled with Student–
Newman–Keuls test. The p-value less than 0.05 were
considered significant.
4. Results
4.1. Effect of REMSD on synaptosomal potential
The DiSC
2
-fluorescence intensity in the synaptosomal
samples prepared from FMC, LPC, REMSD and REC rats
were 0.38 0.05; 0.37 0.05; 0.69 0.05 and 0.49 0.06
fluorescence units/50 mg protein, respectively. The increased
fluorescence value in the REMSD sample reflected relative
(compared to control) depolarized state of the synaptosomes.
The REMSD value was significantly high compared to both
FMC (F = 16.30, d.f. = 4, p < 0.01) and LPC (F = 18.72,
d.f. = 4, p < 0.01) synaptosmes, which however returned to the
baseline level after recovery of REM sleep. The DiSC
2
-
fluorescence intensities of LPC (F = 0.01, d.f. = 4, p = 0.90)
and REC (F = 1.65, d.f. = 4, p = 0.23) were comparable to
FMC (Fig. 1).
4.2. Effect of REMSD on synaptosomal Na–K–ATPase
activity
The Na–K–ATPase activity in synaptosomes of FMC, LPC,
REMSD and REC were 12.60 0.92; 13.16 1.63;
18.86 0.86 and 11.38 1.65 mM Pi released/mg protein h,
respectively. REMSD significantly increased the synaptosomal
Na–K–ATPase activity (F = 23.28, d.f. = 4, p < 0.01) as
compared to the FMC. The enzyme activities in LPC
(F = 0.08, d.f. = 4, p = 0.78) and REC (F = 0.40, d.f. = 4,
p = 0.57) samples were also comparable to that of the FMC
(Fig. 1).
4.3. Effect of NA and its antagonist in vivo
4.3.1. Fluorescence intensity
As mentioned above, REMSD increased the DiSC
2
-
fluorescence intensity; however, the increased intensity was
prevented by intraperitoneal injection of PRZ (0.46 0.08
fluorescence units/50 mg protein; F = 0.79, d.f. = 4, p = 0.39,
compared to FMC) and CLN (0.24 0.02 fluorescence units/
50 mg protein; F = 4.19, d.f. = 4, p = 0.07, compared to FMC)
into the REM sleep deprived rats. Such injections into the FMC
control rats (0.34 0.08 fluorescence units/50 mg protein;
F = 0.09, d.f. = 4, p = 0.77 for PRZ and 0.33 0.05 fluores-
cence units/50 mg protein; F = 0.38, d.f. = 4, p = 0.55 for CLN)
were ineffective in modulating the fluorescence (Fig. 2a).
4.3.2. Na–K–ATPase activity
Intraperitoneal injection of PRZ (14.22 0.55 mMPi
released/mg protein h; F = 2.68, d.f. = 4, p = 0.14, compared
to FMC) and CLN (14.22 0.36 mM Pi released/mg protein h;
F = 2.10, d.f. = 4, p = 0.19, compared to FMC) prevented the
Fig. 1. Percent changes in DiSC
2
-fluorescence intensity and Na–K–ATPase
activity in synaptosomes prepared from LPC, REMSD and REC rat brain as
compared to FMC (taken as 100%) are shown (N = 5). **p < 0.01, significant
as compared to FMC. Abbreviations: as in the text.
G. Das, B.N. Mallick / Neurochemistry International 52 (2008) 734–740736
Page 3
REMSD-induced increase in Na–K–ATPase activity (see
above). However, PRZ (11.71 0.99 mM Pi released/mg pro-
tein h; F = 0.43, d.f. = 4, p = 0.53) or CLN (10.31
1.64 mM Pi released/mg protein h; F = 2.06, d.f. = 4, p =
0.19) alone was ineffective in altering the enzyme activities
in the FMC rats (Fig. 2b).
4.4. Effect of NA and its antagonist in vitro
4.4.1. Fluorescence intensity
Incubation of the FMC synaptosomes with NA (100 mM)
increased DiSC
2
-fluorescence intensity to 0.73 0.07 fluores-
cence units/50 mg protein (F = 15.04, d.f. = 4, p < 0.01)
compared to the untreated control (Ctl) samples (0.38 0.05
fluorescence units/50 mg protein). Further, such NA-induced
increase in DiSC
2
-fluorescence intensity was prevented by pre-
incubating the synaptosomes with PRZ (0.43 0.03 fluores-
cence units/50 mg protein; F = 0.59, d.f. = 4, p = 0.46, compared
to Ctl) but not with PRN (0.66 0.04 fluorescence units/50 mg
protein; F = 14.10, d.f. = 4, p < 0.01, compared to Ctl) (Fig. 3a).
4.4.2. Na–K–ATPase activity
Incubation of the synaptosomes with NA (100 mM)
significantly increased the Na–K–ATPase activity (20.46
1.42 mM Pi released/mg protein h; F = 21.28, d.f. = 4, p <
0.01) compared to its untreated Ctl samples (12.60
0.92 mM Pi released/mg protein h). This NA-induced increase
in the enzyme activity was prevented by pre-incubating
the synaptosomes with PRZ (13.33 1.31 mM Pi released/
mg protein h; F = 0.21, d.f. = 4, p = 0.66, compared to Ctl) but
not by PRN (19.87 1.34 mM Pi released/mg protein h;
F = 19.76, d.f. = 4, p < 0.01, compared to Ctl) (Fig. 3b).
Fig. 2. (a) DiSC
2
-fluorescence intensity in synaptosomal samples prepared
from FMC and REMSD rats with and without i.p. injection of PRZ and CLN
was estimated. The percent changes in the intensities as compared to FMC
(without any injection) value taken as 100%, are shown (N = 5). **p < 0.01,
significant as compared to FMC. Abbreviations: as in the text. (b) Na–K–
ATPase activity in synaptosomes prepared from FMC and REMSD rat brain
with and without i.p. injection of PRZ and CLN was estimated. The percent
changes in activities as compared to FMC (without any injection) value taken as
100%, have been shown (N = 5). **p < 0.01, significant as compared to FMC.
Abbreviations: as in the text.
Fig. 3. (a) DiSC
2
-fluorescence intensity was estimated in samples containing
rat brain synaptosomes (Ctl) and after in vitro incubation of samples with NA
alone as well as in the presence of either PRZ or PRN. The percent changes in
intensity compared to Ctl value taken as 100%, are shown (N = 5). **p < 0.01,
significant as compared to Ctl. Abbreviations: as in the text. (b) Percent changes
in rat brain synaptosomal Na–K–ATPase activities after in vitro incubation of
samples with NA alone as well as in the presence of either PRZ or PRN are
shown (N = 5). **p < 0.01, significant as compared to Ctl. Abbreviations:asin
the text.
G. Das, B.N. Mallick / Neurochemistry International 52 (2008) 734–740 737
Page 4
5. Discussion
Transmembrane potential provides an overview of the state
of affairs of a neuron, which reflects its physiological condition.
It may modulate directly or indirectly a variety of neuronal
functions including signaling, neurotransmitter release and
neurogenesis (Choi, 1988; Hochner et al., 1989; Deisseroth
et al., 2004). The depolarization of neurons brought about either
by an increased influx of positive ions or efflux of the negative
ions, is associated with neuronal excitability (Deisseroth et al.,
2004). It is known that abnormal alteration in the ionic flux may
release apoptotic factors like cytochrome c thereby activating
the caspase cascade which ultimately may lead to its death
(Moon et al., 2005). The Na–K–ATPase plays a key role in
maintaining the transmembrane potential by an efflux of three
Na
+
in exchange for two K
+
influx (Trachtenberg et al., 1981;
Horisberger et al., 1991). Various neurotransmitters also
modulate the neuronal transmembrane potential by regulating
the cellular ion homeostasis either directly by activating ion
channels or indirectly through various signaling molecules. NA
is one such neurotransmitter that has been shown to modulate
membrane depolarization in neurons (Pan et al., 1994).
During REMSD, the level of NA increases in the brain
because (i) the NA-ergic REM-OFF neurons, which normally
cease activity during REM sleep, continue being active
(Mallick et al., 1989); (ii) there is increased synthesis (Sinha
et al., 1973; Majumdar and Mallick, 2003) and decreased
breakdown (Thakkar and Mallick, 1993) of the NA synthesiz-
ing and hydrolyzing enzymes, respectively during REMSD;
(iii) REM sleep is reduced if those neurons are continuously
activated (Singh and Mallick, 1996) or not allowed to cease
activity (Kaur et al., 2004); (iv) NA levels increase after
REMSD (Porkka-Heiskanen et al., 1995); and (v) we have
confirmed that the REMSD associated increased Na–K–
ATPase activity was indeed due to the elevated levels of NA
in the brain (Gulyani and Mallick, 1995; Mallick et al., 2000,
2002). Increased levels of NA bring about various cellular
changes including alterations in biochemical, physiological and
molecular processes, resulting in disturbed homeostasis that has
far reaching effects on the behavior of a subject (Kopp et al.,
1982; Heaney et al., 1999).
The results of this study showed that upon REMSD there
was increased DiSC
2
-fluorescence intensity compared to that of
the FMC. This suggested that the neurons tended to remain in a
relative depolarized state, thereby reducing its threshold for
excitation. Intraperitoneal injection of PRZ and CLN, which
blocks the NA action and release, respectively, prevented the
REMSD-induced depolarization in vivo . Further, incubation of
the synaptosomes with NA in vitro increased the DiSC
2
-
fluorescence, which however, was prevented by PRZ but not by
PRN. Based on these observations we proposed that the
REMSD-induced increased depolarization was mediated by
NA acting through the a1-adrenoceptor. Simultaneously, in the
same synaptosomal sample we observed that REMSD
increased Na–K–ATPase activity and the effect was also
mediated by NA acting on a1-adrenoceptor as reported by us
earlier (Gulyani and Mallick, 1995; Mallick et al., 2000).
Thus, the fact that both Na–K–ATPase activity and
synaptosomal potential were increased after REMSD in the
same sample and both were mediated by NA acting on a1-
adrenoceptor, suggest that the two processes are linked
phenomena. Independent studies have shown that NA
depolarizes a membrane due to a net efflux of the chloride
anions (Lamb and Barna, 1998), thereby activating the voltage-
dependent sodium channels (Takahashi et al., 1999). Thus,
REMSD-induced elevated NA increases intracellular positivity
possibly by an efflux of negative ions resulting in membrane
depolarization. We have shown under similar conditions that
REMSD stimulated the chloride-sensitive Mg-ATPase pump
(Mallick and Gulyani, 1993), which is known to extrude the
negative chloride ions (Shiroya et al., 1989), support the
findings. The REMSD-induced NA mediated membrane
depolarization then activates the voltage-dependent sodium
channels (Catterall, 1992) to cause a net influx of sodium ions,
which also adds to the increase in excitable state of the neuron.
As the intracellular level of Na
+
increases, the neuron tries to
compensate and balance the Na
+
overload inside the cell
(Takahashi et al., 1999) by activating the Na–K–ATPase, which
extrudes three Na
+
in lieu of two K
+
influx (Trachtenberg et al.,
1981; Horisberger et al., 1991). Notwithstanding, both in vivo
and in vitro experiments have shown that the REMSD-induced
increased NA increases the Na–K–ATPase activity (Gulyani
and Mallick, 1995; Mallick et al., 2000). Thus, the NA-induced
stimulation of Na–K–ATPase may be a direct effect or
secondary to efflux of anions. Although it is difficult to
confirm if one mechanism follows the other, we suspect both
the mechanisms exist and they may be activated under different
conditions including various diseases. Therefore, the etiology
and progression of various diseases may be different, though
the symptoms expressed may be similar. Notwithstanding, the
increased Na–K–ATPase activity alters the release of various
neurotransmitters (Vizi et al., 1982) which maybe the cause of
symptoms expressed during REMSD and associated disorders.
The rats were REM sleep deprived by the flower-pot method,
the most preferred method of choice globally for such studies.
Notwithstanding, to rule out the effects of non-specific factors
including stress-induced effects, we carried out other standard
LPC and REC control experiments. Earlier we have shown
under similar conditions that the increased Na–K–ATPase
activity was neither due to increased muscular activity nor due
to movement restriction on the small platform (Gulyani and
Mallick, 1993). Although there may be some loss of non-REM
sleep on the small platform, it is comparable to the LPC rats, at
least after more than 48 h deprivation (Mendelson et al., 1974).
Electrophysiological recordings have shown that this method
indeed induces selective REM sleep loss and the effects were
unlikely due to stress (Porkka-Heiskanen et al., 1995). Besides,
the arguments in favor of the observed effects being specific to
REMSD, as discussed earlier, hold true for this study as well
(Vogel, 1975; Gulyani et al., 2000).
We showed earlier that REMSD increases Na–K–ATPase
activity (Gulyani and Mallick, 1993, 1995) as well as its
expression (Majumdar et al., 2003) on neurons in different
regions of the brain as well as in the whole brain; hence, we
G. Das, B.N. Mallick / Neurochemistry International 52 (2008) 734–740738
Page 5
carried out this study in the whole brain synaptosomes.
However, there are various types of neurons in the brain, e.g.,
excitatory and inhibitory, hence it is possible that the responses
of some neurons may differ and also the effects would depend
on the duration of deprivation. This view may be supported by
the finding in slice preparation that although the dentate gyrus
granule neurons were not affected, the hippocampal CA1
neurons showed reduced firing after REMSD (McDermott
et al., 2003). In related in vivo study it has been shown that the
firing of REM-OFF neurons decreased, while that of REM-ON
neurons increased after REMSD (Mallick et al., 1989), while,
other neurons responded to lower intensity of auditory
stimulation (Mallick et al., 1991). Therefore, it is worth
studying the threshold and excitability of neurons in different
brain areas and specifically on neurochemically identified
neurons. Though this method has been used widely for
synaptosomal potential studies, it has a few limitations. The
DiSC
2
dye might accumulate in different organelles or it might
remain bound to the synaptosomal membrane, thereby affecting
the final fluorescence intensity. However, since we have studied
relative changes in intensity, these are likely to be comparable
in the control samples, unless there is any specific reason to
believe that REMSD alters the synaptosomal membrane
property affecting its interactions with DiSC
2
.
In conclusion, we had proposed that REMSD alters brain
excitability but direct evidence was lacking. Although it was
known that REMSD increases NA that stimulates Na–K–
ATPase activity, we did not know how the enzyme gets
activated. It was also known that intracellular positivity
stimulates the enzyme. In this study, we observed that
indeed the intracellular positivity is increased after REMSD
andthisismediatedbyNAactingthrougha1-adrenoceptor.
Thus, the results of this study confirm and provide
direct evidence in support of our hypothesis that REMSD
increases brain excitability and that is mediated by at
increased level of NA.
Acknowledgements
Research was supported by funding from Indian Council of
Medical Research and University Grants Commission, India.
GD received research fellowship from Council of Scientific and
Industrial Research, India.
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    • "Although PGO waves cannot be recorded in humans, PGO-like field potentials have been recorded in humans (Lim et al., 2007), and increased activity in both lateral geniculate body and occipital cortex has been reported with Positron Emission Tomography (PET) in humans (Hong et al., 1995; Peigneux et al., 2001). It has been shown that REM-D in rats increases the activity of Na-K ATPase enzyme (Gulyani and Mallick, 1993), leading to increased excitability of neurons (Das and Mallick, 2008). The usual inhibitory period observed after repetitive auditory stimulation is shortened after REM-D in evoked potentials (Dewson et al., 1967) and in pontine neurons (Mallick et al., 1991) in cats suggesting maintained excitability to the same stimulation. "
    [Show abstract] [Hide abstract] ABSTRACT: Converging evidence from animal and human studies suggest that rapid eye movement (REM) sleep modulates emotional processing. The aim of the present study was to explore the effects of selective REM sleep deprivation (REM-D) on emotional responses to threatening visual stimuli and their brain correlates using functional magnetic resonance imaging (fMRI). Twenty healthy subjects were randomly assigned to two groups: selective REM-D, by awakening them at each REM sleep onset, or non-rapid eye movement sleep interruptions (NREM-I) as control for potential non-specific effects of awakenings and lack of sleep. In a within-subject design, a visual emotional reactivity task was performed in the scanner before and 24 h after sleep manipulation. Behaviorally, emotional reactivity was enhanced relative to baseline (BL) in the REM deprived group only. In terms of fMRI signal, there was, as expected, an overall decrease in activity in the NREM-I group when subjects performed the task the second time, particularly in regions involved in emotional processing, such as occipital and temporal areas, as well as in the ventrolateral prefrontal cortex, involved in top-down emotion regulation. In contrast, activity in these areas remained the same level or even increased in the REM-D group, compared to their BL level. Taken together, these results suggest that lack of REM sleep in humans is associated with enhanced emotional reactivity, both at behavioral and neural levels, and thus highlight the specific role of REM sleep in regulating the neural substrates for emotional responsiveness.
    Preview · Article · Jun 2012 · Frontiers in Behavioral Neuroscience
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    • "Therefore, it is possible that upon REMSD an increased activity in DR neurons will cause increased Na + concentration inside the neurons. REMSD-induced increased intracellular positivity, a reflection of depolarization of neurons, supports this view [38] . Increased Na + concentration and metabolites inside a cell would cause increased water influx into the neurons due to osmosis, thus resulting in swelling and increased cell size [4,39]. "
    [Show abstract] [Hide abstract] ABSTRACT: This study was carried out to investigate the effect of rapid eye movement sleep (REMS) deprivation (REMSD) on the cytomorphology of the dorsal raphe (DR) neurons and to evaluate the possible role of REMSD-induced increased noradrenalin (NA) in mediating such effects. Rats were REMS deprived by the flowerpot method; free moving normal home cage rats, large platform and post REMS-deprived recovered rats were used as controls. Further, to evaluate if the effects were induced by NA, separate sets of experimental rats were treated (i.p.) with α1-adrenoceptor antagonist, prazosin (PRZ). Histomorphometric analysis of DR neurons in stained brain sections were performed in experimental and control rats; neurons in inferior colliculus (IC) served as anatomical control. The mean size of DR neurons was larger in REMSD group compared to controls, whereas, neurons in the recovered group of rats did not significantly differ than those in the control animals. Further, mean cell size in the post-REMSD PRZ-treated animals was comparable to those in the control groups. IC neurons were not affected by REMSD. REMS loss has been reported to impair several physiological, behavioral and cellular processes. The mean size of the DR neurons was larger in the REMS deprived group of rats than those in the control groups; however, in the REMS deprived and prazosin treated rats the size was comparable to the normal rats. These results showed that REMSD induced increase in DR neuronal size was mediated by NA acting on α1-adrenoceptor. The findings suggest that the sizes of DR neurons are sensitive to REMSD, which if not compensated could lead to neurodegeneration and associated disorders including memory loss and Alzheimer's disease.
    Full-text · Article · Oct 2010 · Behavioral and Brain Functions
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    • "Disturbed or loss of REM sleep is witnessed in several psychosomatic pathological conditions like schizophrenia, epilepsy, mood disorder, memory loss, etc. (Benca, 2001; Monti and Monti, 2005; Walker and Stickgold, 2006; Gottesmann and Gottesmann, 2007 ) while it is also correlated with an increase in neuronal excitability (Mallick et al., 1989Mallick et al., , 1994 Das and Mallick, 2007). It has been shown that Na-K-ATPase directly modulates neuronal transmembrane potential (Trachtenberg et al., 1981; Horisberger et al., 1991). "
    [Show abstract] [Hide abstract] ABSTRACT: Rapid eye movement (REM) sleep deprivation elevates noradrenaline level, which upon acting on alpha1-adrenoceptors increases Na-K-ATPase activity; however, the detailed intracellular mechanism of action was unknown. Since membrane integrity is crucial for maintaining Na-K-ATPase activity as well as ionic exchange and noradrenaline affects membrane lipid-peroxidation, we proposed that the deprivation might modulate membrane lipid-peroxidation, which would modulate intracellular ionic concentration and thereby increase Na-K-ATPase activity. Hence, in this in vivo and in vitro study, rats were deprived of REM sleep for 4 days by the flowerpot method and suitable control experiments were conducted. The deprivation simultaneously decreased membrane lipid-peroxidation as well as increased Na-K-ATPase activity by its dephosphorylation and all the effects were induced by noradrenaline. Further, in vitro experiments showed that hydrogen peroxide (H(2)O(2))-induced enhanced lipid-peroxidation increased synaptosomal calcium (Ca(2+))-influx, which was also prevented by noradrenaline and nifidipine, an L-type Ca(2+)-channel blocker. Additionally, both nifidipine and cyclopiazonic acid, which have opposite effects on intracellular Ca(2+)-concentration, prevented deprivation induced increased Na-K-ATPase activity. We propose that REM sleep deprivation elevates noradrenaline level in the brain that acting on alpha1-adrenoceptor simultaneously reduces membrane lipid-peroxidation but activates phospholipase-C, resulting in closure of L-type Ca(2+)-channel and releasing membrane bound Ca(2+); the latter then dephosphorylates Na-K-ATPase, the active form, causing its increased activity.
    Full-text · Article · Jun 2008 · Neuroscience
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