The Effects of Nicotine on Spatial Learning and Memory and DREAM Protein Expression on REM Sleep Deprived Rat Hippocampus

THE EFFECTS OF
NICOTINE TREATMENT ON
SPATIAL LEARNING AND MEMORY
AND DREAM PROTEIN EXPRESSION IN
REM-SLEEP DEPRIVED RAT
HIPPOCAMPUS
MOHAMMAD JAMEL MOHD ALWAI
SCHOOL OF HEALTH SCIENCES,
UNIVERSITI SAINS MALAYSIA HEALTH CAMPUS

Introduction
• Sleep is critical for the maintenance of health and support of
life.
• Two types of sleep; rapid eye movement (REM) and non-REM
sleep (Campbell, 2009)
• REM sleep is important for learning and memory formation in
the hippocampus (Stickgold & Walker, 2007)
• REM sleep deprivation can affect hippocampal synaptic
plasticity – vital for learning acquisition and memory
consolidation (Yang et al., 2008)

DREAM Protein
• Downstream Regulatory Antagonistic Modulator (DREAM)
protein involved in learning and memory processes.
• Acts as negative regulator of the key memory factor cAMP-
response element binding (CREB) protein in a Ca2+-dependent
manner.
• DREAM gene knockout facilitates CREB-dependent
transcription and markedly enhances learning and synaptic
plasticity with improved cognition.
(Fontan-Lozano et al., 2009)

Nicotine (C10H14N2)
• Derived from plants of family
Solanaceae, from genus Nicotiana spp.
(Chase et al., 2003)
• An agonist for nicotinic acetylcholine
receptor (Poorthuis et al., 2009).
• Acute treatment of nicotine has been
shown to prevent impairment of
memory and synaptic plasticity of CA1
region of hippocampus due to REM
sleep-deprivation (Aleisa et al., 2011)
Figure 1: Chemical
structure of nicotine

Rationale of Study
To study how nicotine treatment can prevent
impairment of learning and memory induced
by REM sleep deprivation.

Research Question
Whether administration of nicotine can
prevent impairment of spatial learning and
memory and synaptic plasticity through
modulation of DREAM protein expression in
REM sleep-deprived rat hippocampus.

General Objective
To determine the effects of nicotine treatment
on spatial learning and memory and
DREAM protein expression in REM sleep-
deprived rat hippocampus.

Specific Objectives
• To determine the effects of nicotine (nicotinic
acetylcholine receptor agonist) treatment on
spatial learning and memory performance on
REM sleep-deprived rats.
• To determine the effects of nicotine (nicotinic
acetylcholine receptor agonist) treatment on
DREAM protein expression in REM sleep-
deprived rat hippocampus.

Drug Administration
• Nicotine (1mg/kg) (Aleisa et al., 2006) was
injected subcutaneously every 12h during
72h REM sleep-deprivation.
• The dose given is known to produce
nicotine blood levels similar to those of
chronic smokers (Le Houezec et al., 1993)

REM sleep-deprivation
• REM sleep deprivation used modified
inverted flowerpot method (Siran et al.,
2009).
• Non-REMsd rats were placed in normal
propylene cages.

Morris Water Maze
• Circular pool filled within 20cm off the top
of wall with water (20-22ºC).
• Water were made opaque by skim milk
powder
• Platform (Ø 12cm, white Plexiglas).

Morris Water Maze
• A camera was mounted on top of the
maze and connected to a PC with MWM
software.
• Before performing the task, rats were
placed on the platform for
10 s to familiarise it-self with the
maze.
• Rats were trained to find platform from
4 different directions around the edge
of the pool for every day for 5 days.
Figure 2: Morris Water
Maze procedure

Morris Water Maze
• The time (s) of escape latency were recorded to evaluate
spatial learning and memory performance.
• If rats failed to find platform in 60s, they were placed on
the platform; latency were recorded as 60s.
• Surrounding objects/visual objects were fixed to serve
as cues for the rats to locate the platform.
• Probe test was performed at day 6 without the platform
to measure time rats spent in original platform position
(target quadrant)

Immunohistochemistry analysis
• After 24h MWM, rats were sacrificed via
overdose injection of sodium pentobarbitone.
• Brains were dissected out, cryoprotected in
sucrose 20% overnight.
• Brain sections were cut 40 µm thin using
cryostat and hippocampus region collected as
free-floating sections in PBS.

• Sections were rinsed with Tris-buffered saline (TBS) and
incubated with primary antibodies for DREAM protein for 48h.
• Then, the sections were incubated with biotinylated secondary
antibody for 1h.
• Sections were then reacted with Avidin-biotin complex (ABC)
and stained with diaminobenzidine and hydrogen peroxide
until brown colour is seen
• Section then were mounted on slides, air-dried, dehydrated
and put cover slip on it.

Body weight gain
Comparison between groups in body weight gain during dry and wet condition.
Dry condition Wet condition
Body weight gain was reduced in both REMsd and REMsd+N
groups during wet condition.
RESULTS:

Food Intake
Comparison in food intake during dry and wet condition for all groups.
There was no significant difference in food intake during dry
and wet condition for all groups.

Comparison in water intake during dry and wet condition for all
groups.*, p < 0.01, compared between dry and wet conditions
Water intake was reduced in REM sleep deprivation groups
during wet condition.
Water intake

Escape latency
Escape latency time for all groups during MWM 5 days trial.
Within days, escape latency time for all groups were
reduced however not significant difference between groups
during 5 days trial.

Percentage of time spent in target quadrant
Percentage of time spent in target quadrant in all groups.
#, p < 0.05, compared between C+N and REMsd
??, p < 0.05, compared between C and REMsd
There was significantly least time spent in target quadrant for
REMsd group.

DREAM protein expression
DREAM protein expression of A) control group (C); B) nicotine-treated
control (C+N); C) sleep-deprived group (REMsd); and D) nicotine-treated
sleep-deprived group (REMsd+N).
(CA1= CA1 area of hippocampus; CA3= CA3 area of the hippocampus; DG= dentate
gyrus)

DISCUSSIONS
• Data for food and water intake and body weight
– REMsd and REMsd+N rats showed constant food intake but
significant decrease in body weight and water intake during wet
condition
– Similar result found in previous study in our lab (Siran et al.,
2014)
– Decrease in body weight with constant food intake represent
negative energy state by a number of changes to physiological
systems
– Sleep will usually limit metabolic rate i.e. there was an increase
in energy expenditure
• These results suggests the accomplishment of induction
of REM sleep-deprivation in rats in this study

Morris Water Maze
• Result show gradual decrease of escape latency recorded
by all groups within 5 days of MWM trial.
• C+N group has the fastest time in finding the platform
especially in days 3 to 5 compared to other groups.
– However a study (Alzoubi et al., 2006) reported
nicotine treatment may have no effects on normal
subjects.
– Our results could suggest that rats experienced mild
stress during some part of the experiment.

Probe test
• REMsd group has the least percentage of time spent in target
quadrant.
– This supports our understanding that REM sleep-deprived rats
have impaired some of their learning and memory processes.
• However the nicotine treatment showed some of the beneficial
effects by abolishing this effect (as shown by REMsd+N groups)
• Meanwhile the percentage of time spent in target quadrant in
REMsd+N was found to be not significantly different compared to C
and C+N groups.
• Transdermal nicotine treatment has also been showed to abolish
mild cognitive impairment in Alzheimer patients (Roh & Evins, 2012)

• Preliminary results showed that DREAM protein was
expressed in all regions of hippocampus in all
groups.
– This propose that DREAM protein could be
involved in spatial and learning processes in this
study.
• However, the mechanism how DREAM protein
modulated the spatial learning and memory
processes are still unclear.

CONCLUSION
• This study shows that REM sleep deprivation
partially affected spatial learning and memory
performance.
• Treatment of nicotine has improved this effect.
• The contribution of DREAM protein modulation
in this effect is still unclear and required
further study to elucidate the mechanism.

LIMITATION
• Time constraints
– Sleep deprivation using wide platform cannot be
done.
• Technical problems
– Immunohistochemistry study cannot be finished
– Comparison of DREAM protein expression
between groups cannot be seen.

FUTURE DIRECTIONS
• Further studies are needed to link between the
expression of DREAM protein and spatial
learning and memory performance. This could
be done by
– Quantifying DREAM protein expression in the
hippocampus and compare between groups.
– Quantifying another protein which involved in
learning and memory such as CREB and BDNF
protein.
– Prolonging the period of MWM study by addition of
reverse test after the probe test where platform is
positioned at opposing quadrant than that of original
position and trial is done for another 5 days.

REFERENCES
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(2011). Acute nicotine treatment prevents REM sleep deprivation-induced learning and memory impairment in
rat. Hippocampus, 21(8), 899-909.
• Alzoubi, K. H., Aleisa, A. M., Gerges, N. Z., & Alkadhi, K. A. (2006). Nicotine reverses adult-onset
hypothyroidism-induced impairment of learning and memory: Behavioral and electrophysiological studies.
Journal of Neuroscience Research, 84(5), 944-953.
• Campbell, I. G. (2009). EEG Recording and Analysis for Sleep Research. Current Protocols in Neuroscience, 49.
• Chase, M. W., Knapp, S., Cox, A. V., Clarkson, J. J., Butsko, Y., Joseph, J., Savolainen, V., Parokonny, A. S. (2003).
Molecular Systematics, GISH and the Origin of Hybrid Taxa in Nicotiana (Solanaceae). Annals of Botany, 92(1),
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• Fontán-Lozano, Á., Romero-Granados, R., del-Pozo-Martín, Y., Suárez-Pereira, I., Delgado-García, J. M.,
Penninger, J. M., & Carrión, Á. M. (2009). Lack of DREAM Protein Enhances Learning and Memory and Slows Brain
Aging. Current Biology, 19(1), 54-60.
• Le Houezec, J., Jacob, P., III, & Benowitz, N. L. (1993). A clinical pharmacological study of subcutaneous nicotine.
European Journal of Clinical Pharmacology, 44(3), 225-230.
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Journal of Physiology and Biochemistry, 70(4), 877-889.
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The Effects of Nicotine on Spatial Learning and Memory and DREAM Protein Expression on REM Sleep Deprived Rat Hippocampus

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