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PLANT-DERIVED NANOPARTICLE TREATMENT WITH COCC 30C
AMELIORATES ATTENTION AND MOTOR ABILITIES
IN SLEEP-DEPRIVED RATS
S. ZUBEDAT,
a
Y. FREED,
c
Y. ESHED,
c
A. CYMERBLIT-SABBA,
a
A. RITTER,
a
M. NACHMANI,
a
R. HARUSH,
d
S. AGA-MIZRACHI
a
AND A. AVITAL
a,b
*
a
The Rappaport Faculty of Medicine, Behavioural Neuroscience Lab,
Technion – Israel Institute of Technology, Haifa, Israel
b
Emek Medical Center, Afula, Israel
c
The International Institute for Homeopathy Research, Hod
Hasharon, Israel
d
Yezrel Valley College, Afula, Israel
Abstract—Sleep is an essential physiological process that
underlies crucial cognitive functions as well as emotional
reactivity. Thus, sleep deprivation (SD) may exert various
deleterious effects. In this study, we aimed to examine
the adverse behavioral and hormonal effects of SD and
a potential treatment with Plant-derived nanoparticle
treatment – cocc 30c. The study was a 4-arm trial with
randomization and double-blinding of verum and placebo
treatments. SD was induced by using the Multiple Platform
Method for 48 h. The effects of SD were evaluated behavior-
ally (pre-pulse inhibition (PPI), startle response and rotor-
rod) at baseline as well as at 6, 12, 24 h, and 14 days post
deprivation. cocc 30c treatment was administrated Per Os
every three hours starting immediately after baseline tests
and for a period of 24 h. On day 14, blood samples were
taken and serum levels of corticosterone, testosterone,
serotonin and leptin were tested. We found that cocc 30c
improved PPI 12 and 24 h post deprivation, likewise, cocc
30c improved motor learning. On day 14 SD led to increased
startle response that was ameliorated by cocc 30c. Likewise,
SD led to increased levels of corticosterone and serotonin
while decreasing testosterone and leptin. Interestingly,
cocc 30c treatment has moderated these hormonal altera-
tions. We conclude that the treatment with cocc 30c
recovers both short-term behavioral and the long-term hor-
monal modulations following SD. Ó2013 IBRO. Published
by Elsevier Ltd. All rights reserved.
Key words: attention, nanoparticles, cocc 30c, motor
learning, sleep deprivation, hormones.
INTRODUCTION
Sleep deprivation (SD)
In humans, sleep is an essential physiological process
which, when deprived, may exert deleterious effects. In
the literature, SD is divided into three categories: long-
term total SD (>45 h); short-term total SD (645 h); and
partial SD (sleep restriction to <7 h/24 h) (Durmer and
Dinges, 2005). Considering the above, acute total SD of
24–48 h was previously shown to impair the
performance in both attention (Blagrove et al., 1995;
Bocca and Denise, 2006; Kendall et al., 2006) and
working memory (Wimmer et al., 1992; Smith et al.,
2002).
Positron Emission Tomography (PET) scans
confirmed that 24 h of SD decreases glucose
metabolism and synaptic activity in the prefrontal cortex,
an area involved in attention processes, as well as in
dorsal and ventral thalami (Thomas et al., 2000; Kato
et al., 2000). Kato et al. (2000) showed that SD led to
an increase in blood pressure and a decrease in muscle
sympathetic nerve activity. Furthermore, Van leeuwen
et al. (2009) found that SD increases the risk of
cardiovascular diseases by augmenting pro-
inflammatory responses. Other studies have also
showed results of severe symptoms such as irritability,
fatigue, hallucinations, and delusions (Orzel-
Gryglewska, 2010). Overall, SD in humans has been
found to impair attention (Alhola and Polo-Kantola,
2007; McCoy and Strecker, 2011), cognitive functions,
and behavioral performance (Curcio et al., 2006). While
SD’s deteriorating behavioral effects are suggested to
be normally recovered (Schwierin et al., 1999;Faraut
et al., 2012), the duration needed for this recovery was
found to be dependent on the deprivation paradigm.
Specifically, longer SD requires a longer normal
recovery period (McCoy and Strecker, 2011).
TREATMENTS OF SD
Treatment of SD commonly involves psycho-stimulants
such as caffeine, which may restore attention. However
they are not effective when evaluating cognitive tasks,
decision-making or motor activities (Killgore et al., 2012).
Evidence of the efficiency of homeopathic treatment
with cocc 30c given to patients suffering from SD has
been accumulated in our clinic (unpublished data).
Patients who received cocc 30c remedy reported an
improvement in their ability to sleep, reduced anxiety/
0306-4522/13 $36.00 Ó2013 IBRO. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuroscience.2013.08.021
*Corresponding author. Address: Behavioural Neuroscience Lab, The
Rappaport Faculty of Medicine, Gutwirth Building, Technion – Israel
Institute of Technology, Haifa 32000, Israel. Tel: +972-50-6503361;
fax: +972-4-6495231.
E-mail addresses: Avital@tx.technion.ac.il,Avitalavi@hotmail.com
(A. Avital).
These authors contributed equally to this work.
Abbreviations: 5-HT, 5-hydroxytryptamine; ANOVA, analysis of
variance; D1R, dopamine receptor1; ITI, Inter-Trial-Interval; MPM,
Multiple Platform Method; PPI, pre-pulse inhibition; SD, sleep
deprivation.
Neuroscience 253 (2013) 1–8
1
irritability as well as improvement in their cognitive
capabilities. These results were based upon clinical
observations and patients’ reports, thus requiring
systematic validation.
Homeopathy treatment, although partially disputable,
is prevalent both in clinics and research (Lucertini et al.,
2007; Mishra et al., 2011). Accumulating data indicate
detectable effects of different types of remedies (Bell
et al., 2011a), and biological effect of homeopathic
treatment on physiological measures of sleep (Bell
et al., 2011b). Furthermore, in 2012, the Swiss
Government published a health technology assessment
of homeopathy that found strong evidence supporting
homeopathic treatment at least in some medical
treatments (Bornho
¨ft and Matthiessen, 2012).
Specifically, already in 1927 Boericke depict that plant
source material Cocculus has a potential for reversing
the effects of sleep loss (Boericke, 1927).
Finally, new basic science data suggest that the
source materials of the medicine do survive and persist
in nanoparticulate form across homeopathically
prepared potencies, including cocc 30c, as a function of
the unique manufacturing processes (Chikramane et al.,
2010, 2012). Biological effects may be mediated in part
by endogenous adaptive responses (Bell and Koithan,
2012). The ability of nano-forms of plant-derived
nanoparticles to cross the blood–brain-barrier was
previously suggested to be mediated by nano-silica or
nano-silicon vehicles and biological amplifiers that would
be augmenting the Cocculus nanoparticles (Demangeat,
2010; Dhawan et al., 2011; Mathew et al., 2012).
ANIMAL MODELS
Previous animal studied have demonstrated the biological
effects of various homeopathic treatments for SD (Ruiz-
Vega et al., 2002, 2005). Nunes Junior et al. (1994)
used the Multiple Platform Method (MPM) as suggested
animal model to induce SD. This method was practiced
in several studies (Suchecki et al., 1998; Suchecki and
Tufik, 2000; Yang et al., 2010) and proved to be
efficient in inducing Rapid-eye-movement (REM)
deprived rats, generating less stress vis-a
`-vis other
acceptable SD methods (Rechtschaffen and Bergmann,
2002; Machado et al., 2006). In rats, chronic SD was
shown to cause death after 16–21 days from the onset
of deprivation. In comparison, food deprivation was
shown to cause death after 17–19 days (Orzel-
Gryglewska, 2010). On the other hand, acute SD was
not observed to cause destructive effects either on cells,
or vital organs (Orzel-Gryglewska, 2010). However, it
increases energy outlay (Martins et al., 2010), with a
tendency to decrease both leptin (Rosa Neto et al.,
2010) and the anabolic testosterone (Wu et al., 2011;
Dattilo et al., 2012). Moreover, SD increases serum
serotonin (Hipolide et al., 2005) as well as its
extracellular concentrations in the hippocampus. It
elevates corticosterone level (Bodosi et al., 2004; Tiba
et al., 2008; Galvao Mde et al., 2009; Martins et al.,
2010; Rosa Neto et al., 2010; Wu et al., 2011), while
this elevation is independent of the stress response
(Galvao Mde et al., 2009; Mongrain et al., 2010) and is
suggested to occur due to changes in circadian rhythm
(Tartar et al., 2009) and metabolic homeostasis (Dattilo
et al., 2012).
When comparing an animal model to humans, one
must take into consideration physiological as well as
brain growth trajectories, especially when rat and human
maturation cannot be compared as linear timeline
development (Erecinska et al., 2004; Quinn, 2005).
HYPOTHESIS AND AIMS
A systematic human study of the effects of SD is ethically
limited. Moreover, controlling, mediating and moderating
potential artifact variables are especially difficult when
examining the long-term effects of SD on humans’
cognitive functioning. Thus, it is customary to use an
animal model in order to investigate the long-term
effects of SD. Similar to humans, in rats sleep is an
important physiological process regarded as a basic
need for functioning and survival (Everson, 1995). Many
studies have proposed different deprivation paradigms,
including partial chronic (Machado et al., 2006) or acute
(Suchecki et al., 1998;Schwierin et al., 1999;Suchecki
and Tufik, 2000) deprivations, with similar effectiveness.
While considering treatment of SD, normal
spontaneous recovery must be taken into account
(Schwierin et al., 1999;Orzel-Gryglewska, 2010). Our
current study aims are to explore the effects of acute
SD during early adulthood, focusing on short-term
behavioral effects as well as on long-term hormonal
modulations. Specifically, we aimed to examine the
short- (post 1-, 6-, 12- and 24-h) and long-term (post
14 days) effects of acute 48 h SD on fatigue, attention,
and motor learning. Moreover, we aimed to examine the
outcomes of cocc 30c treatment on both behavioral and
hormonal modulations following SD.
EXPERIMENTAL PROCEDURES
Animals
Forty-four male Wistar rats (weighing between 200 and
220 gr) were purchased from Harlan (Jerusalem, Israel)
and were given 7 days of acclimation in the institutional
animal housing facility. Rats were housed four per cage
(30
L
30
W
18
H
cm). Room temperature was
maintained at 23 ± 1 °C with 67% humidity at 12:12-
day/night cycle (lights on at 0600). Food and water
access were allowed ad libitum. This study was
conducted in strict accordance with the
recommendations of the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health.
The protocol was approved by the Institutional Animal
Care and Use Committee. All efforts were made to
minimize animal suffering.
Procedure
Rats were randomly assigned into four groups: Naive
(n= 12), Naive rats that were treated with cocc 30c
(Naive Treated; n= 12), sleep deprived (SD; n= 16),
and sleep deprived + Treatment (SD treated; n= 16).
2 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
Starting on Postnatal day (PND) 70 rats were subjected to
SD for 48 h, using MPM (method as described below)
(Suchecki and Tufik, 2000). cocc 30c or placebo was
given Per Os starting immediately after baseline tests
(i.e. 1 h post SD), and subsequently every 3 h during
24 h post SD. One hour after SD, prior to treatment, rats
were tested in the startle box for baseline evaluation.
Additional re-tests were held at 6, 12 and 24 h, as well
as at 14 days post SD. Pre-pulse inhibition (PPI) was
examined at 12 and 24 h. Motor learning test in the
Rotor-rod was performed starting 48 h post-SD (for total
of 4 days of training). Rats were handled prior to any
manipulation. A description of the procedure is shown in
Fig.1.
MPM. This method is based on the muscle atonia
characteristics of REM sleep. In short, this method
[modified from (Suchecki and Tufik, 2000)] includes
placing the animal on top of a narrow platform located
inside a water tank that allows the animal to lie down.
As muscle atonia occurs, the rat falls into the water and
is deprived of REM sleep.
In the current study, the water tank (120 cm in
diameter, 60 cm in height) contained 30 narrow
platforms (6.5 cm in diameter) placed inside. Sixteen
animals (in each round) were placed on top of the
platforms, allowing them to move around. The tank was
filled with water (6 cm) until 1 cm below the platform,
thus allowing the rat to climb back in case of falling into
the water. Exposure to the MPM began at 0800, and
animals remained inside the tank for 48 h. In order to
verify the effectiveness of MPM, rats were videotaped
by an infra-red Ikegami camera. Room temperature was
maintained at 23 ± 1 °C. Food and water were provided
ad libitum. All rats accomplished the entire study (no
deaths occurred).
cocc 30c treatment. cocc 30c is a homeopathic
remedy made of highly diluted tincture of Anamirta
cocculus seeds powder, first prepared and published by
Hahnemann in 1821 (Hahnemann, 1821). The plant
originates from the coast of Malabar, India, and Ceylon.
The tincture is prepared by macerating the powdered
seeds. The Cocculus preparation: The Cocculus was
prepared in ‘helios pharmacy (UK)’. It was sent in a
bottle containing 99% ethanol and went through 40
succussions in 90% ethanol, by manual shaking. Sixty
globules were wetted in the solution and dried for
several minutes. Following this procedure, cocc 30c
made from sucrose immersed in a solution of Cocculus
indicus 30c (40 mg dissolved in 1 ml tap water), was
administered Per Os every three hours immediately
after baseline tests for a period of 24 h. Placebo
treatment was 1% sucrose solution similar to the
hedonic effect caused by the sweet taste of cocc 30c.
The experiment was carried out in a random double-
blind design i.e. Experimenter 1 (Dr. Freed) prepared
the cocc 30c or placebo treatments in two identical
tubes (labeled ‘‘A’’ or ‘‘B’’) and delivered to the principal
investigator (Dr. Avital) the tubes on the day of
experiment. The experimenters (all the authors except
Dr. Avital and Freed) got the labeled tubes. Across total
number of rats, a randomized assignment to one of four
groups was made until accomplishing a total assignment
of 16 rats in the treated groups and 12 in the Naive and
Naive Treated rats. cocc 30c or placebo treatments
were revealed after the results analysis.
Behavioral tests
PPI and startle response. The test is held in a
ventilated soundproof box (Campden instruments, UK)
and aims to examine Startle response as well as the
function of the sensorimotor gating.
The session (a total of 90 trials) begins with three min
acclimation period with a 57-dB background white noise
level that is delivered continuously throughout the test
session. To evaluate the startle response, each of the
first ten trials consist of a single 40-ms 120-dB ‘‘pulse
alone’’ startle stimuli (Inter-Trial-Interval (ITI) 1 min). The
rest of the 80 trials (10 s ITI) consist of random delivery
of: 10 ‘‘no stimuli’’ trials, during which no stimuli are
delivered, fourteen ‘‘pre’’ stimuli (at 59, 61, 65, 69, 73,
78 or 85 dB), and 56 ‘‘pre-pulse’’ trials that include a
single 120 dB pulse preceded (80 ms interval) by a
20 ms pre pulse of 2, 4, 8, 12, 16, 21 or 28 dB above
background (i.e., 59, 61, 65, 69, 73, 78 or 85 dB).
Finally, PPI was calculated as the percent of the
habituated response as follows: [100 (max response
to ‘‘pre-pulse’’ trial/max response to ‘‘pulse alone’’
trial 100)] (Avital et al., 2011; Ram et al., 2013).
Rotor-rod. The rotor-rod (San Diego instruments, San
Diego, CA, USA) apparatus is used to assess motor
functions, motor learning, coordination, and equilibrium.
It is made of black lusterless Perspex, comprised of a
rotating rod with four lanes separated by opaque black
Perspex. The apparatus contains a sawdust cabin
(45.7 cm height) for safe landing and automatic
recording of latencies (in sec.) via red-beams system.
Each learning session consists of four daily trials, on
four consecutive days. Rats are acclimated to the
apparatus for 30 s on the first day. Each trial starts at
five rpm for 15 s with constant acceleration at 0.1 rpm
per second (max speed 50 rpm after 460 s).
Corticosterone, testosterone, serotonin and leptin
evaluation
In order to avoid circadian variability, all samples were
collected between 1100 and 1200, when plasma
hormones’ concentration is relatively low. Blood
samples were taken immediately after decapitation and
centrifuged (2000gat 4 °C for 20 min), serum was
collected and stored at 80 °C until assayed. Serum
corticosterone and testosterone levels were assessed
using commercial enzyme-linked immunosorbent assay
(ELISA) kits (AssayPro, St. Charles, MO, USA)
according to the manufacturer’s instructions.
Serum leptin was quantified using specific enzyme
immunoassay (Genese
Ò
, Brazil). Serotonin
concentration was determined using commercial
S. Zubedat et al. / Neuroscience 253 (2013) 1–8 3
enzyme assay kit (Diagnostic products Corp., Los
Angeles, CA, USA) (Wu et al., 2011).
Statistical analysis
A Bonferroni multiple correction analysis was performed
in order to avoid possible confounds due to the
substantial amount of testing. Data were analyzed for
statistical significance using an analysis of variance
(ANOVA) for mixed design, with group as between-
subject factor and test timing or pre-pulse intensity as
within-subject factor. In order to further explore the main
effects, we used a one-way ANOVA followed by a Post-
Hoc Tukey test. A result was considered significant
when P< 0.05. All tests were calculated as two-tailed
using SPSS V17.0. Results are presented as
means ± standard error of the means (SEM).
RESULTS
A Bonferroni multiple correction analysis was performed
in order to avoid possible confounds and the substantial
amount of testing after the SD that may bound to affect
the recovery sleep. The analysis yielded no effect, thus,
it excludes the possible confound that may affect the
sleep recovery.
Startle response
In order to examine short- and long-term progression in
startle response (Fig. 2), an ANOVA for mixed design
was carried-out with group as between-subject factor
and test time as within-subject factor. The results
indicate a significant effect for test time
[F(4,17) = 39.96, P< 0.0001] as well as between
groups [F(3,20) = 7.2, P< 0.002]. Post-hoc Tukey
tests indicate a significant decrease in startle response
for both SD and SD-treated groups compared with naive
rats, at baseline (P< 0.0001), post-6 h (P< 0.0001)
and post-12 h (P< 0.01). Twenty-four hours post SD all
groups showed similar and low startle response that
probably reflects habituation and/or floor effect. Finally,
14 days post SD, while naive- and SD-treated rats
showed a recovery (from the alleged habituation) to the
naive rats’ startle response, the SD rats showed a long-
term increased startle response compared with naive
(P< 0.007), naive-treated (P< 0.0001) and SD-treated
rats (P< 0.0001).
PPI
PPI 12 h after SD (Fig. 3): a significant difference was
found along various pre-intensities [F(6, 47) = 102.97,
P< 0.0001] as well as between the groups
[F(3,52) = 4.53, P< 0.007]. Overall, a clear tendency
of impaired PPI was observed following SD.
Interestingly, the treatment with cocc 30c led to a
beneficial effect on PPI performance. Specifically, a
significant effect was found at pre-intensity 61 dB
between SD-treated and SD (P< 0.0001), naive
(P< 0.044) and naive-treated (P< 0.001) rats.
Regarding pre-intensities 65 dB and 69 dB cocc 30c led
to improved PPI compared with SD (P< 0.001;
P< 0.025, respectively).
PPI 24 h post SD (Fig. 3): similarly to 12 h post-SD
test, a significant effect was observed along different
pre-intensities [F(6,47) = 115.97, P< 0.0001] as well
as between the groups [F(3, 52) = 11.47, P< 0.0001].
The treatment with cocc 30c clearly led to elevated PPI
performance. In particular, SD-treated rats have
exhibited a significant increase at pre-intensity 61 dB,
compared with both SD (P< 0.0001) and naive
(P< 0.01) rats. Following SD, the treatment cocc 30c
led to improved PPI at pre-intensities 65 dB
(P< 0.0001), 69 dB (P< 0.0001), 73 dB (P< 0.001),
78 dB (P< 0.001) and 85 dB (P< 0.005), compared
with SD group.
Rotor-rod
A significant motor learning effect (Fig. 4) was
found during 4 days of training [F(3, 132) = 49.72,
Fig. 1. A schematic description of the experimental procedure. A description of experimental time line and postnatal days, along which the exposure
to sleep deprivation, treatment, behavioral and hormonal examinations took place.
4 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
P< 0.0001] with significant difference between the
groups [F(3,134) = 39.74, P< 0.0001]. Moreover, a
significant effect was found for the group day
interaction [F(9,392) = 2.43, P< 0.011]. Post-hoc
Tukey test indicated a significantly enhanced
performance in the SD-treated group, compared with
SD (P< 0.0001), naive (P< 0.0001) and naive-treated
(P< 0.028), on the first day of motor learning. This
enhanced performance for the treated SD was also
significant (P< 0.0001) on the second day compared
with SD, naive and naive-treated. On the third day SD-
treated performed better compared with naive
(P< 0.004) and SD (P< 0.0001) groups. This
tendency remains on the fourth day of motor learning,
as the SD-treated rats performed better than SD rats
(P< 0.0001).
Corticosterone
A significant group effect on corticosterone serum
(Fig. 5A) levels [F(3,54) = 50.72, P< 0.0001] was
observed. Post-hoc Tukey test indicated a significant
elevation in corticosterone following SD (P< 0.0001)
compared with naive and naive-treated groups.
However, the treatment with cocc 30c significantly
moderated this elevation toward (P< 0.0001; compared
with SD group).
Testosterone
A significant group effect on testosterone serum (Fig. 5B)
level [F(3,51) = 13.35, P< 0.0001] was observed. Post-
hoc Tukey test indicated a significant decrease in
testosterone level following SD (P< 0.0001), while cocc
30c treatment significantly restored this decrease
(P< 0.013; compared with SD group).
Serotonin
A significant group effect on serotonin serum (Fig. 5C)
level [F(3,51) = 23.46, P< 0.0001] was observed.
Post-hoc Tukey test indicated a significant elevation
following SD (P< 0.0001), while cocc 30c treatment
moderated the observed elevation (P< 0.0001;
compared with SD group).
Leptin
A significant group effect on leptin serum (Fig. 5D) level
[F(3,51) = 118.15, P< 0.0001] was observed. Post-
hoc Tukey test indicated a significant decrease in leptin
level following SD (P< 0.0001), while cocc 30c
treatment significantly repaired the latter decrease to a
moderated level (P< 0.0001; compared with SD group).
DISCUSSION
Attentional and motor reactivity
Both treated and untreated SD groups showed hypo-
responsiveness to the startle stimulus. Though naive
rats showed habituation along the four time points that
were examined (i.e. baseline, 6, 12 and 24 h post-SD),
the lack of this habituation in the SD groups may be
attributed to floor effect. Fourteen days post SD, all
groups showed a recovery from the alleged habituation,
similar to the naive group. However, the SD group
Fig. 2. Startle reflex. A significant decrease in startle response was observed in both SD and SD-treated groups compared with naive rats, at
baseline, post-6 h and post-12 h. Fourteen days post SD, while naive- and SD-treated rats showed a recovery (from the alleged habituation) to the
naive rats startle response, the SD rats showed a long-term increased startle response compared with naive with SD-treated rats (
⁄⁄⁄
P< 0.0001;
⁄⁄
P< 0.01).
Fig. 3. Pre-pulse inhibition (PPI). The treatment with cocc 30c led to
a beneficial effect on PPI performance measured both 12- and 24-h
post SD (
⁄⁄⁄
P< 0.0001;
⁄⁄
P< 0.001;
⁄
P< 0.005;
#
P< 0.025).
S. Zubedat et al. / Neuroscience 253 (2013) 1–8 5
showed a significant increased startle response.
Considering the noise during the MPM procedure in
which 16 rats stayed together in the same arena, one
may postulate that the effects of ambient noise in the
different environments can explain the hypo-
responsiveness to the startle stimulus (Baldwin et al.,
2006). This elevation in startle response observed in all
groups, may reflect the extensive test procedure that
the rats underwent, together with evidence that startle
response is suggested to be age-dependent (Weiss
et al., 2001).
Previously it has been shown that SD in humans
impairs attention ability (Alhola and Polo-Kantola, 2007;
McCoy and Strecker, 2011). Specifically, acute SD
(such as total 24–48-h deprivation) was previously
shown to impair the performance in attention tasks
(Blagrove et al., 1995; Kendall et al., 2006; Bocca and
Denise, 2006). Thus, we aimed to test the sensorimotor
gating utilizing the PPI test, which relates to attention
processes (Avital et al., 2011; Ram et al., 2013). SD led
to impaired PPI ability 12 h post deprivation.
Surprisingly, the treated rats showed an immediate
effect, with superior PPI ability compared with all other
groups. Twenty-four hours post SD, though there was
no significant difference in PPI performance between
the control and the SD groups (presumably due to
spontaneous recovery from SD), yet the SD-treated rats
showed a better PPI performance compared with all
other groups. The striatum is considered to be the
relevant region for sensorimotor gating (Moore et al.,
2006). Indeed, Lim et al. (2011) have recently found a
significant decrease in dopamine receptor1 (D1R), no
change in D2R, and a significant increase in D3R
binding in the striatum, following SD. This pattern was
not observed following stress, thus suggesting to be a
specific remodeling of dopaminergic circuits after SD.
Considering dopamine as the core neurotransmitter
involved in attention processes in both the prefrontal
cortex and striatum (Moore et al., 2006; Kumari et al.,
2008; Molina et al., 2009), we postulate that the
beneficial effect of cocc 30c on PPI performance,
immediately following SD, may be associated to the
increased binding to D3R in the striatum.
Fig. 5. Hormonal serum levels. Fourteen days after the exposure to sleep deprivation, a significant increase in both corticosterone (A) and serotonin
(B) serum level was observed in the SD group. However, the treatment with cocc 30c had a long-term beneficial effect, manifested in a significant
decrease of both corticosterone and serotonin. Moreover, sleep deprivation led to decreased serum levels of Testosterone (C) and Leptin (D).
Interestingly, cocc 30c treatment recovered these decrements (
⁄⁄⁄
P< 0.0001;
⁄⁄
P< 0.013).
Fig. 4. Motor learning in the rotor-rod test. The treatment with cocc
30c led to a better motor learning ability (starting 48 h post SD) across
all 4-days learning SD (
⁄⁄⁄
P< 0.0001).
6 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
Taken together, the behavioral tests have indicated
significant short-term effects of SD. The treatment with
cocc 30c remedy seems to improve these short-term
deteriorating effects
Hormonal modulations
In order to explore whether SD has a long-term ‘‘covert’’
hormonal effects while the ‘‘overt’’ behavior effects are
recovered, we examined corticosterone, testosterone,
serotonin and leptin serum levels, 14 days post SD. The
notion on ‘‘covert’’ hormonal and ‘‘overt’’ behavioral
effects of SD is supported by Lopez-Rodriguez et al.
(2003) report that extracellular concentration of
serotonin remained high at the end of SD recovery day
period, though displaying normal amount of sleep.
Following SD, corticosterone and serotonin serum
levels were elevated and the treatment with cocc 30c
recovered these elevations. Moreover, testosterone and
leptin decreased following SD, and cocc 30c treatment
moderated this decline. Our findings are in line with a
previous study (Wu et al., 2011), which reported a
reduction of serum testosterone, and elevated levels of
serotonin and corticosterone, following SD. Considering
the inverse secretion relation between testosterone and
5-hydroxytryptamine (5-HT) (Frungieri et al., 2002), it is
plausible that the reduction in testosterone level is due
to 5-HT inhibition of testosterone production, or vice
versa. Similarly to our 48-h SD effect on corticosterone
and leptin levels, it was previously found that 96 h of SD
led to increased corticosterone as well as decreased
leptin serum levels (Rosa Neto et al., 2010). Moreover,
Koban and Swinson (2005) showed that leptin
decreased after SD and remained low following twenty
days of recovery.
CONCLUSIONS
Taken together, the treatment with cocc 30c seems to
restore the deteriorating effects of 48 h of SD on
attention and motor learning abilities. Examining the
long-term effects of SD, cocc 30c dramatically
recovered the hormonal alterations observed.
Bioavailability and biological activity of nano-forms of
any material in general and specifically of cocc 30c,
suggest its therapeutic potential intriguing, as CNS
access across the blood–brain barrier is readily possible
for the small sized nanoparticles that were already
shown present in homeopathic medicines (Chikramane
et al., 2010). Furthermore, recent study by Barve and
Chuaughule (2013) have shown that succussions can
mechanically reduce the initial particle size of plant
extracts in homeopathic manufacturing processes into
the very small nanoparticle range (e.g., approximately
13 nm). This observation further supports the possibility
of blood–brain access.
REFERENCES
Alhola P, Polo-Kantola P (2007) Sleep deprivation: impact on
cognitive performance. Neuropsychiatr Dis Treat 3:553–567.
Avital A, Dolev T, Aga-Mizrachi S, Zubedat S (2011) Environmental
enrichment preceding early adulthood methylphenidate treatment
leads to long term increase of corticosterone and testosterone in
the rat. PLoS One 6:e22059.
Baldwin AL, Primeau RL, Johnson WE (2006) Effect of noise on the
morphology of the intestinal mucosa in laboratory rats. J Am
Assoc Lab Anim Sci 45:74–82.
Barve R, Chaughule R (2013) Size-dependent in vivo/in vitro results
of homoeopathic herbal extracts. J Nanostruct Chem 3:18.
Bell IR, Koithan M (2012) A model for homeopathic remedy effects:
low dose nanoparticles, allostatic cross-adaptation, and time-
dependent sensitization in a complex adaptive system. BMC
Complement Altern Med 12:191.
Bell IR, Brooks AJ, Howerter A, Jackson N, Schwartz GE (2011a)
Short-term effects of repeated olfactory administration of
homeopathic sulphur or pulsatilla on electroencephalographic
alpha power in healthy young adults. Homeopathy 100:203–211.
Bell IR, Howerter A, Jackson N, Aickin M, Baldwin CM, Bootzin RR
(2011b) Effects of homeopathic medicines on polysomnographic
sleep of young adults with histories of coffee-related insomnia.
Sleep Med 12:505–511.
Blagrove M, Alexander C, Horne JA (1995) The effects of chronic
sleep reduction on the performance of cognitive tasks sensitive to
sleep deprivation. Appl Cogn Psychol 9:21–40.
Bocca ML, Denise P (2006) Total sleep deprivation effect on
disengagement of spatial attention as assessed by saccadic eye
movements. Clin Neurophysiol 117:894–899.
Bodosi B, Gardi J, Hajdu I, Szentirmai E, Obal Jr F, Krueger JM
(2004) Rhythms of ghrelin, leptin, and sleep in rats: effects of the
normal diurnal cycle, restricted feeding, and sleep deprivation. Am
J Physiol Regul Integr Comp Physiol 287:R1071–R1079.
Boericke W (1927) Pocket manual of homeopathic materia
medica. Santa Rosa, CA: Boericke and Tafel, Inc..
Bornho
¨ft G, Matthiessen Peter F (2012) Homeopathy in healthcare –
effectiveness, appropriateness, safety, costs. Berlin, Heidelberg:
Springer. http://rd.springer.com/book/10.1007/978-3-642-20638-
2/page/1.
Chikramane PS, Suresh AK, Bellare JR, Kane SG (2010) Extreme
homeopathic dilutions retain starting materials: a nanoparticulate
perspective. Homeopathy 99:231–242.
Chikramane PS, Kalita D, Suresh AK, Kane SG, Bellare JR (2012)
Why extreme dilutions reach non-zero asymptotes: a
nanoparticulate hypothesis based on froth flotation. Langmuir
28:15864–15875.
Curcio G, Ferrara M, De Gennaro L (2006) Sleep loss, learning
capacity and academic performance. Sleep Med Rev
10:323–337.
Dattilo M, Antunes HK, Medeiros A, Monico-Neto M, Souza Hde S,
Lee KS, Tufik S, de Mello MT (2012) Paradoxical sleep
deprivation induces muscle atrophy. Muscle Nerve 45:431–433.
Demangeat JL (2010) NMR relaxation evidence for solute-induced
nanosized superstructures in ultramolecular aqueous dilutions of
silica-lactose. J Mol Liquids 155:71–79.
Dhawan S, Kapil R, Singh B (2011) Formulation development and
systematic optimization of solid lipid nanoparticles of quercetin for
improved brain delivery. J Pharm Pharmacol 63(3):342–345.
Durmer JS, Dinges DF (2005) Neurocognitive consequences of sleep
deprivation. Semin Neurol 25:117–129.
Erecinska M, Cherian S, Silver IA (2004) Energy metabolism in
mammalian brain during development. Prog Neurobiol
73:397–445.
Everson CA (1995) Functional consequences of sustained sleep
deprivation in the rat. Behav Brain Res 69:43–54.
Faraut B, Boudjeltia KZ, Vanhamme L, Kerkhofs M (2012) Immune,
inflammatory and cardiovascular consequences of sleep
restriction and recovery. Sleep Med Rev 16:137–149.
Frungieri MB, Zitta K, Pignataro OP, Gonzalez-Calvar SI, Calandra RS
(2002) Interactions between testicular serotoninergic,
catecholaminergic, and corticotropin-releasing hormone systems
modulating cAMP and testosterone production in the golden
hamster. Neuroendocrinology 76:35–46.
S. Zubedat et al. / Neuroscience 253 (2013) 1–8 7
Galvao Mde O, Sinigaglia-Coimbra R, Kawakami SE, Tufik S,
Suchecki D (2009) Paradoxical sleep deprivation activates
hypothalamic nuclei that regulate food intake and stress
response. Psychoneuroendocrinology 34:1176–1183.
Hahnemann S (1821) Materia medica pura. Jain Publishers (P) Ltd.
pp. 491.
Hipolide DC, Moreira KM, Barlow KB, Wilson AA, Nobrega JN, Tufik S
(2005) Distinct effects of sleep deprivation on binding to
norepinephrine and serotonin transporters in rat brain. Prog
Neuropsychopharmacol Biol Psychiatry 29:297–303.
Kato M, Phillips BG, Sigurdsson G, Narkiewicz K, Pesek CA, Somers VK
(2000) Effects of sleep deprivation on neural circulatory control.
Hypertension 35:1173–1175.
Kendall AP, Kautz MA, Russo MB, Killgore WD (2006) Effects of
sleep deprivation on lateral visual attention. Int J Neurosci
116:1125–1138.
Killgore WD, Grugle NL, Balkin TJ (2012) Gambling when sleep
deprived: don’t bet on stimulants. Chronobiol Int 29:43–54.
Koban M, Swinson KL (2005) Chronic REM-sleep deprivation of rats
elevates metabolic rate and increases UCP1 gene expression in
brown adipose tissue. Am J Physiol Endocrinol Metab
289:E68–E74.
Kumari V, Fannon D, Geyer MA, Premkumar P, Antonova E,
Simmons A, Kuipers E (2008) Cortical grey matter volume and
sensorimotor gating in schizophrenia. Cortex 44:1206–1214.
Lim MM, Xu J, Holtzman DM, Mach RH (2011) Sleep deprivation
differentially affects dopamine receptor subtypes in mouse
striatum. Neuroreport 22:489–493.
Lopez-Rodriguez F, Wilson CL, Maidment NT, Poland RE, Engel J
(2003) Total sleep deprivation increases extracellular serotonin in
the rat hippocampus. Neuroscience 121:523–530.
Lucertini M, Mirante N, Casagrande M, Trivelloni P, Lugli V (2007)
The effect of cinnarizine and cocculus indicus on simulator
sickness. Physiol Behav 91:180–190.
Machado RB, Suchecki D, Tufik S (2006) Comparison of the sleep
pattern throughout a protocol of chronic sleep restriction induced
by two methods of paradoxical sleep deprivation. Brain Res Bull
70:213–220.
Martins PJ, Marques MS, Tufik S, D’Almeida V (2010) Orexin
activation precedes increased NPY expression, hyperphagia, and
metabolic changes in response to sleep deprivation. Am J Physiol
Endocrinol Metab 298:E726–E734.
Mathew A, Fukuda T, Nagaoka Y, Hasumura T, Morimoto H, Yoshida Y,
Maekawa T, Venugopal K, Kumar DS (2012) Curcumin loaded-
PLGA nanoparticles conjugated with Tet-1 peptide for potential use
in Alzheimer’s disease. PLoS One 7(3):e32616.
McCoy JG, Strecker RE (2011) The cognitive cost of sleep lost.
Neurobiol Learn Mem 96:564–582.
Mishra N, Muraleedharan KC, Paranjpe AS, Munta DK, Singh H,
Nayak C (2011) An exploratory study on scientific investigations in
homeopathy using medical analyzer. J Altern Complement Med
17:705–710.
Molina V, Montes C, Tamayo P, Villa R, Osuna MI, Perez J, Sancho C,
Lopez-Albuquerque T, Cardoso A, Castellano O, Lopez DE (2009)
Correlation between prepulse inhibition and cortical perfusion during
an attentional test in schizophrenia. A pilot study. Prog
Neuropsychopharmacol Biol Psychiatry 33:53–61.
Mongrain V, Hernandez SA, Pradervand S, Dorsaz S, Curie T,
Hagiwara G, Gip P, Heller HC, Franken P (2010) Separating the
contribution of glucocorticoids and wakefulness to the molecular
and electrophysiological correlates of sleep homeostasis. Sleep
33:1147–1157.
Moore H, Jentsch JD, Ghajarnia M, Geyer MA, Grace AA (2006)
A neurobehavioral systems analysis of adult rats exposed
to methylazoxymethanol acetate on E17: implications for
the neuropathology of schizophrenia. Biol Psychiatry 60:
253–264.
Nunes Junior GP, Tufik S, Nobrega JN (1994) Autoradiographic
analysis of D1 and D2 dopaminergic receptors in rat brain after
paradoxical sleep deprivation. Brain Res Bull 34:453–456.
Orzeł-Gryglewska J (2010) Consequences of sleep deprivation. Int J
Occup Med Environ Health 23:95–114.
Quinn R (2005) Comparing rat’s to human’s age: how old is my rat in
people years? Nutrition 21:775–777.
Ram E, Raphaeli S, Avital A (2013) Prepubertal chronic stress and
ketamine administration to rats as a neurodevelopmental model of
schizophrenia symptomatology. Int J Neuropsychopharmacol (in
press).
Rechtschaffen A, Bergmann BM (2002) Sleep deprivation in the rat:
an update of the 1989 paper. Sleep 25:18–24.
Rosa Neto JC, Lira FS, Venancio DP, Cunha CA, Oyama LM,
Pimentel GD, Tufik S, Oller do Nascimento CM, Santos RV, de
Mello MT (2010) Sleep deprivation affects inflammatory marker
expression in adipose tissue. Lipids Health Dis 9:125.
Ruiz-Vega G, Perez-Ordaz L, Leon-Hueramo O, Cruz-Vazquez E,
Sanchez-Diaz N (2002) Comparative effect of Coffea cruda
potencies on rats. Homeopathy 91:80–84.
Ruiz-Vega G, Poitevin B, Perez-Ordaz L (2005) Histamine at high
dilution reduces spectral density in delta band in sleeping rats.
Homeopathy 94:86–91.
Schwierin B, Borbe
´ly AA, Tobler I (1999) Prolonged effects of 24-h
total sleep deprivation on sleep and sleep EEG in the rat.
Neurosci Lett. 261:61–64.
Smith ME, McEvoy LK, Gevins A (2002) The impact of moderate
sleep loss on neurophysiologic signals during working-memory
task performance. Sleep 25:784–794.
Suchecki D, Tufik S (2000) Social stability attenuates the stress in the
modified multiple platform method for paradoxical sleep
deprivation in the rat. Physiol Behav 68:309–316.
Suchecki D, Lobo LL, Hipolide DC, Tufik S (1998) Increased ACTH
and corticosterone secretion induced by different methods of
paradoxical sleep deprivation. J Sleep Res 7:276–281.
Tartar JL, Ward CP, Cordeira JW, Legare SL, Blanchette AJ,
McCarley RW, Strecker RE (2009) Experimental sleep
fragmentation and sleep deprivation in rats increases
exploration in an open field test of anxiety while increasing
plasma corticosterone levels. Behav Brain Res 197:450–453.
Thomas M, Sing H, Belenky G, Holcomb H, Mayberg H, Dannals R,
Wagner H, Thorne D, Popp K, Rowland L, Welsh A, Balwinski S,
Redmond D (2000) Neural basis of alertness and cognitive
performance impairments during sleepiness. I. Effects of 24 h of
sleep deprivation on waking human regional brain activity. J Sleep
Res 9:335–352.
Tiba PA, Oliveira MG, Rossi VC, Tufik S, Suchecki D (2008)
Glucocorticoids are not responsible for paradoxical sleep
deprivation-induced memory impairments. Sleep 31:505–515.
van Leeuwen WM, Lehto M, Karisola P, Lindholm H, Luukkonen R,
Sallinen M, Harma M, Porkka-Heiskanen T, Alenius H (2009)
Sleep restriction increases the risk of developing cardiovascular
diseases by augmenting proinflammatory responses through
IL-17 and CRP. PLoS One 4:e4589.
Weiss IC, Domeney AM, Moreau JL, Russig H, Feldon J (2001)
Dissociation between the effects of pre-weaning and/or post-
weaning social isolation on prepulse inhibition and latent inhibition
in adult Sprague–Dawley rats. Behav Brain Res 121:207–218.
Wimmer F, Hoffmann RF, Bonato RA, Moffitt AR (1992) The effects
of sleep deprivation on divergent thinking and attention
processes. J Sleep Res 1:223–230.
Wu JL, Wu RS, Yang JG, Huang CC, Chen KB, Fang KH, Tsai HD
(2011) Effects of sleep deprivation on serum testosterone
concentrations in the rat. Neurosci Lett 494:124–129.
Yang RH, Wang WT, Hou XH, Hu SJ, Chen JY (2010) Ionic
mechanisms of the effects of sleep deprivation on excitability in
hippocampal pyramidal neurons. Brain Res 1343:135–142.
(Accepted 13 August 2013)
(Available online 22 August 2013)
8 S. Zubedat et al. / Neuroscience 253 (2013) 1–8