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
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, 3,30-
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 platformand recoverycontrolswere carried outinaddition tofree-moving control. Thefluorescence
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: DiSC2; 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
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
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
(Kushidaetal., 1989; Gulyanietal., 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
leveland lower isthe threshold ofresponsivenessofthe 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
Available online at www.sciencedirect.com
Neurochemistry International 52 (2008) 734–740
* Corresponding author. Tel.: +91 11 26704522; fax: +91 11 26717558.
E-mail address: email@example.com (B.N. Mallick).
0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
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
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 ofNA inthe presence and absence ofitsantagoniston
Noradrenaline (NA); a1-adrenoceptor antagonist, prazosin
(PRZ); b-adrenoceptor antagonist, propranolol (PRN); a2-
adrenoceptor agonist, clonidine (CLN); Na–K–ATPase inhi-
bitor, ouabain; mono-cationic
diethylthiacarbocyanine iodide (DiSC2)and dimethylsulfoxide
(DMSO) were procured from Sigma–Aldrich, USA. All other
chemicals were of analytical grade.
All the animal experimental protocols were approved by the
(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 fivedifferent sets, each havingone 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
minimizetheuseofnumberofratsand finally,data from45 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 quicklyremovedand
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 NaHCO3, 1 mM MgCl2?6H2O,
1.2 mM Na2HPO4, 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
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
DiSC2is 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 DiSC2movement inside the cells
is directly proportional to the intracellular negativity (Wang
et al., 2001). The anions inside the synaptosomes bind to DiSC2
and consequently quench its fluorescence. Hence, greater the
intracellular negativity more was the DiSC2 quenching,
resulting in reduced net fluorescence intensity. Thus, intensity
of fluorescence was inversely proportional to the intracellular
anion concentration and as a corollary increased DiSC2-
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
by measuring the intensity of the cationic fluorescent probe
DiSC2as described previously by Perkinton and Sihra (1998)
and was expressed as fluorescence units/50 mg synaptosomes.
In brief, the DiSC2stock 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
10 min at 37 8C, following which the reaction was terminated
byadditionofice-cold HEPES buffer andthevolumewas made
up to 2 ml. The DiSC2-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,
2.4. Estimation of Na–K–ATPase activity
(Gulyani and Mallick, 1993). Synaptosomes (40 mg) were
incubated with the reaction buffer containing 100 mM NaCl,
20 mM KCl, 5 mM MgCl2, 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.
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
4.1. Effect of REMSD on synaptosomal potential
The DiSC2-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 DiSC2-
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
The Na–K–ATPase activity in synaptosomes of FMC, LPC,
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
12.60 ? 0.92; 13.16 ? 1.63;
4.3. Effect of NA and its antagonist in vivo
4.3.1. Fluorescence intensity
As mentioned above, REMSD increased the DiSC2-
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 mgprotein; F = 0.38, d.f. = 4,p = 0.55 forCLN)
were ineffective in modulating the fluorescence (Fig. 2a).
4.3.2. Na–K–ATPase activity
Intraperitoneal injection of PRZ (14.22 ? 0.55 mM Pi
released/mg protein h; F = 2.68, d.f. = 4, p = 0.14, compared
toFMC)andCLN(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 DiSC2-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
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)
1.64 mM Pi released/mg protein h;
0.19) alone was ineffective in altering the enzyme activities
in the FMC rats (Fig. 2b).
orCLN (10.31 ?
F = 2.06, d.f. = 4,p =
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 DiSC2-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 DiSC2-fluorescence intensity was prevented by pre-
incubating the synaptosomes with PRZ (0.43 ? 0.03 fluores-
cenceunits/50 mgprotein;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,comparedtoCtl)(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) DiSC2-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
changesin activitiesascomparedto FMC(withoutany 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) DiSC2-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,
significantas comparedto 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: as in
G. Das, B.N. Mallick/Neurochemistry International 52 (2008) 734–740737
Transmembrane potential provides an overview of the state
It may modulate directly or indirectly a variety of neuronal
functions including signaling, neurotransmitter release and
neurogenesis (Choi, 1988; Hochner et al., 1989; Deisseroth
by an increased influx of positive ions or efflux of the negative
ions, is associated with neuronal excitability (Deisseroth et al.,
2004).Itis known that abnormalalterationin 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
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
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 DiSC2-
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 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
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
DiSC2dye might accumulate in different organelles or it might
the final fluorescence intensity. However, sincewe 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 DiSC2.
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
and this is mediated by NA acting through a1-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.
Research was supported by funding from Indian Council of
Medical Research and University Grants Commission, India.
GD receivedresearch fellowship from Councilof Scientificand
Industrial Research, India.
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