BMX Drosophila

Effects of Bacopa monnieri Supplementation on the Learning and Short-Term Memory Retention
of Sleep-Deprived Drosophila melanogaster
Bradley Ashley G. Ong

Maria Clarice N. Villanueva
An Undergraduate Thesis
Submitted to the Department of Biology
College of Arts and Sciences
University of the Philippines Manila
Padre Faura, Manila
In partial fulfillment of the requirements

For the degree of
Bachelor of Science in Biology
May 2016
Effects of Bacopa monnieri…
Ong and Villanueva, 2016
Department of Biology
Padre Faura, Manila
Announcement of the
Undergraduate Thesis Presentation
BRADLEY ASHLEY GUE ONG

and
MARIA CLARICE NUQUI VILLANUEVA
Entitled
EFFECTS OF Bacopa monnieri SUPPLEMENTATION
ON LEARNING AND SHORT-TERM MEMORY RETENTION
OF SLEEP-DEPRIVED Drosophila melanogaster
For the degree of

Bachelor of Science in Biology
1:00 P.M., 13 April 2016

Room 115, Rizal Hall
THESIS ADVISER THESIS CO-ADVISER

Miriam P. de Vera, Ph.D. Paul Mark B. Medina, Ph.D.
Associate Professor Associate Professor
Department of Biology Department of Biochemistry
College of Arts and Sciences College of Medicine
University of the Philippines Manila University of the Philippines Manila
THESIS READER THESIS READER

Melody Anne B. Ocampo, M.S. Ma. Cristina A. Bordallo, M.P.H.
Assistant Professor Senior Lecturer
Department of Biology Department of Biology
College of Arts and Sciences College of Arts and Sciences
University of the Philippines Manila University of the Philippines Manila
Endorsed by: Authorized by:
Miriam P. de Vera, Ph.D. Elena M. Ragragio, MA Sci. Educ.

Chair Chair
Thesis Committee Department of Biology
ii
Department of Biology
Padre Faura, Manila
ENDORSEMENT
The thesis attached hereto, EFFECTS OF Bacopa monnieri SUPPLEMENTATION

ON LEARNING AND SHORT-TERM MEMORY RETENTION OF SLEEP-
DEPRIVED Drosophila melanogaster, prepared and submitted by Bradley Ashley G. Ong
and Maria Clarice N. Villanueva in partial fulfillment of the requirements for the degree of
Bachelor of Science in Biology, was successfully defended on 13 April 2016.
MIRIAM P. DE VERA, Ph.D. PAUL MARK B. MEDINA, Ph.D.

Thesis Adviser Thesis Co-Adviser
MELODY ANNE B. OCAMPO, M.S. MA. CRISTINA A. BORDALLO, M.P.H.

Thesis Reader Thesis Reader
This undergraduate thesis is hereby officially accepted as partial fulfillment of the

requirements for the degree of Bachelor of Science in Biology.
ELENA M. RAGRAGIO, MA Sci. Educ. LEONARDO R. ESTACIO Jr., Ph.D.

Chair Dean
Department of Biology College of Arts and Sciences
iii
BIOGRAPHICAL DATA
Bradley Ashley G. Ong
I. Personal Information
Full Name: Bradley Ashley Gue Ong
Nickname: Brad, Ash
Birthday: 22 February 1995
27 Doña Aurora Street Araneta Village
Permanent Address: Potrero, Malabon City
1476 NCR, Philippines
Mobile Number: +639175272823
Electronic Mail Address: bradleyashleyong@gmail.com
Father’s Name: Joselito Reyes Co Ong
Mother’s Name: Vicky Gue Ong
II. Educational Background

Primary Jubilee Christian Academy, Quezon City, Philippines
Secondary Jubilee Christian Academy, Quezon City, Philippines

(First Honorable Mention)
Collegiate University of the Philippines Manila, Manila, Philippines
(Bachelor of Science in Biology, Magna cum laude standing)
III. Collegiate Organizations

Member (2012-2014) and Vice President for External Affairs (2015-2016), Cerulean
Member (2013-2014), Lifebox
Member (2013–2014), UP One Earth (UPOE)
Member (2013–2014), P.R.O. Internals (2014-2015) and Vice President (2015-2016)
U.P. Biological Sciences Society (BSS)
Member (2013–2016), U.P. Biology Major’s Association (BIOMAS)
Member (2013-2016), Biology Majors Alliance of the Philippines (BMAP)
Externals Head (2014-2015) and Member (2015-2016), The Hope Project (THP)
Member (2014-2016), Registration Volunteer Corporation (RVC)
iv
Member (2014-2016), U.P. Manila Biorhythm

Managing Editor (2015-2016), Aninag Editorial Board
Member (2015-2016), Phi Kappa Phi (PKP)
Member (2015-2016), Ugnayan ng Pahinungod
Member (2016-2017), Phi Sigma Biological Sciences Honor Society
BIOGRAPHICAL DATA
v
Maria Clarice N. Villanueva
I. Personal Information
Full Name: Maria Clarice Nuqui Villanueva
Nickname: Claire, Cla
Birthday: 18 June 1996
Purok 6, Old Road, San Jose Village,
Permanent Address: City of San Fernando, Pampanga
2000 Region III, Philippines
Mobile Number: +639358663547
Electronic Mail Address: clrcvillanueva@yahoo.com
Father’s Name: Alvin Santos Villanueva
Mother’s Name: Imelda Nuqui Villanueva
II. Educational Background

Primary San Fernando Elementary School, Pampanga, Philippines
(Fourth Honorable Mention)
Secondary Pampanga High School, Pampanga, Philippines
(Fourth Honorable Mention)
Collegiate University of the Philippines Manila, Manila, Philippines
(Bachelor of Science in Biology)
III. Collegiate Organizations

Member (2012-2013), U.P. Biological Sciences Society (BSS)
Member (2013-2016) and Vice President for Finance (2014-2016), UP Manila
Indayog Dance Varsity
vi
ACKNOWLEDGEMENT
The authors are formally indebted to a small group of individuals who made this
undergraduate thesis experience extremely endurable and worthwhile. Heartfelt gratitude is given
to the following:
 Dr. Miriam de Vera, the adviser, for her essential and valuable corrections, gentle prodding and
germane suggestions throughout the entire course of this endeavour,
 Dr. Paul Medina, the co-adviser, for his thorough guidance, unfaltering patience, surprising
amicability and generosity, in both time and resources, that provided a unique first-hand
perspective on the workings and potentials of our study,
 Prof. Melody Ocampo and Mrs. Cristina Bordallo, the readers, for their positive criticisms
and out-of-the-box insights leading to the expansion and improvement of this work,
 Mr. Ben Lopez, an L.U. IV student of the College of Medicine, for essentially and voluntarily
giving a crash course on Drosophila breeding, from food medium preparation to specimen
handling,
 Ms. Kim Cochon, presently an instructor at the Department of Epidemiology and Biostatistics,
College of Public Health, for her invaluable assistance during the statistical analysis phase of the
research,
 National Institutes of Health, for generously providing funds for the completion of our study
from the first day until the very last,
 PCUM Robotics Trading, for supplying professional-level schematic drawings and the
construction of the improvised assay apparatus and sleep deprivation machine employed in this
study,
 the Ong and Villanueva families, together with their friends: Ashley, Chanel, Hanna, Lea,
Nathea, and Shaira, for the emotional, financial and moral support during this 10-month struggle,
and
 the Lord Almighty, for the strength and everything in between.
Bradley Ashley G. Ong Maria Clarice N. Villanueva
vii
TABLE OF CONTENTS
Title Page ................................................................................................................................ …..i

Announcement ............................................................................................................................. ii
Endorsement ............................................................................................................................... iii
Biographical Data ....................................................................................................................... iv
Acknowledgement ..................................................................................................................... vii
Table of Contents ...................................................................................................................... viii
List of Tables .............................................................................................................................. ix
List of Figures ...............................................................................................................................x
List of Appendices ...................................................................................................................... xi
Abstract ...................................................................................................................................... xii
Introduction ...................................................................................................................................1
Review of Related Literature ........................................................................................................4
Materials and Methods ................................................................................................................12
Results .........................................................................................................................................19
Discussion ...................................................................................................................................22
Conclusion ..................................................................................................................................27
Recommendations .......................................................................................................................28
Literature Cited ...........................................................................................................................29
Tables ..........................................................................................................................................36
Figures.........................................................................................................................................39
Appendix 1. Media......................................................................................................................43
Appendix 2. T-maze Setup for Aversive Phototaxic Suppression Assay (APS) ........................44
Appendix 3. Sleep Nullifying Apparatus (SNAP) ......................................................................45
Appendix 4. Methodology Flowchart .........................................................................................46
Appendix 5. Analysis of Variance (ANOVA) and Post Hoc Analaysis .....................................47
viii
LIST OF TABLES
Table 1. Difference on the Quinines Sensitivity Index of both non-slpD and slpD Drosophila….36
Table 2. Average Pass Rates for Learning and Memory Retention of non-slpD Drosophila…. 37
Table 3. Average Pass Rates for Learning and Memory Retention of slpD Drosophila…….....38
ix
LIST OF FIGURES
Figure 1. Survival rate of Drosophila melanogaster on the difference of 10-fold concentration

of Bacopa monnieri supplement over the course of seven days……………….….…..39
Figure 2. Comparison of the effect of velocity stressed caused by mechanical stimulation of the
machine on the learning and short-term memory retention of D. melanogaster……...39
Figure 3. Standardized average pass rates for learning and memory retention of non-sleep-
deprived and sleep-deprived D. melanogaster ………………………………….........40
Figure 4. Pass-Fail Map for Learning and STM retention of fruit flies………………………....41
Figure 5. Mean score of photonegative choices by non-sleep-deprived D. melanogaster on the
learning phase and short-term memory retention of different treatment groups….….42
Figure 6. Mean score of photonegative choices by sleep-deprived D. melanogaster on the
learning phase and short-term memory retention of different
treatment groups.………………………………………………………….…….….....42
x
LIST OF APPENDICES
Appendix 1. Media.................................……………………………………………………………43
Appendix 1a. Media Synthesis……………………………….……………………….........……...43
Appendix 1b. Summary of the Bacopa monnieri Memory Enhancing Supplement………...…..43
Appendix 2. T-Maze Setup for Aversive Phototaxis Suppression Assay…………………….….…..44
Appendix 3. Sleep Nullifying Apparatus………..…………………………………………….....45
Appendix 4. Methodology flowchart…………………………………………………………….46
Appendix 5. ANOVA Results and Post Hoc Results…………………………………………….47

Appendix 5a. Two way ANOVA results for Velocity Stress Test…………………………….47
Appendix 5b. Two way ANOVA results for the Learning Phase……………………………….47
Appendix 5c. Pairwise Comparisons of Marginal Linear Predictions between the control and
treatment groups for the Learning Phase……………………………………………………..47
Appendix 5d. Pairwise Comparisons of Marginal Linear Predictions for the Learning Phase
Using Bonferroni Method…………………………..............…………………………………….48
Appendix 5e. Two way ANOVA results for the Memory Retention Phase..................................49
Appendix 5f. Pairwise Comparisons of Marginal Linear Predictions between the control and
treatment groups for the Memory Retention Phase…....................................................................49
Appendix 5g. Pairwise Comparisons of Marginal Linear Predictions for the Memory Retention
Phase Using Bonferroni Method………………………………………………………………….49
Appendix 5h. Analysis of Covariance (ANCOVA) results between Average Pass Rates of
Learning and Memory Retention…………………………………………………………………50
xi
ABSTRACT
Aversive phototaxic suppression (APS) is a behavioral assay in which photopositive fruit

flies (Drosophila melanogaster) are trained to avoid light using quinine as an aversive stimulus.
This conditioned behavior has been found to be impaired by sleep deprivation in D. melanogaster.
This study tested whether supplementation with Bacopa monnieri can counter this impairment.
Three sublethal and effective concentrations of B. monnieri supplement were given to test groups
of non-sleep-deprived (slp-) and sleep-deprived (slp+) flies subjected to the APS assay. The extent
of learning and short-term memory (STM) retention were based on average pass rates (APR)
defined as the number of trials that each fly exhibited a photonegative response before and after
conditioning. Results showed that B. monnieri supplement improved the APR of both slp- and slp+
flies as compared to the unsupplemented flies. These effects were found to be dose-dependent
from the low to the mid-level (4.62x10-4g/mL to 4.62x10-3g/mL) concentrations of the supplement
on D. melanogaster learning and STM retention.
xii
INTRODUCTION
Background of the Study
Nootropics, commonly known as memory enhancers, are compounds that act on cognitive
functions. These type of supplements are purportedly used to treat cognitive deficits such as in
Alzheimer’s disease, Huntington’s disease, and attention deficit hyperactivity disorder or ADHD
(Barkley, 2006). The claims of the manufacturers of these dietary supplements posed a problem
due to the lack of verifiable therapeutic effects that is verified by industrial entities (Hathcock,
2001). Nonetheless, these off-label and non-prescription supplements have been increasingly
commonplace for non-medical situations like shift-works, jet lags and active military personnel
(Sahakian and Morein-Zamir, 2007). These situations are related to sleep deprivation and fatigue
leading to decrements in cognition as well as in psychomotor vigilance response speed or the
reaction time to sustained-attention to visual stimulus (Belenky et al., 2003). Sleep deprivation can
lead to significant changes in hippocampal activity during episodic memory encoding, and results
in impaired subsequent retention (Yoo, 2007).
The fruit fly, Drosophila melanogaster, has long been utilized as a neurophysiological-
behavioral model to understand various human cognitive deficits for many reasons. The organism
can be reliably trained to learn and remember association between stimuli through both classical
and operant conditioning (Tully and Quinn, 1985).
Statement of the Problem
Does Bacopa monnieri supplementation have an effect on the learning capacities and short-
term memory (STM) retention of non-sleep-deprived and sleep-deprived Drosophila
melanogaster?
1
Research Objectives
The general objective of this study is to test the potential effects of B. monnieri supplement
on the learning capacities and STM retention in non-sleep-deprived and sleep-deprived D.
melanogaster subjected to the aversive phototaxic suppression (APS) assay. The specific
objectives are: (1) to demonstrate if the photopositive behavior of D melanogaster can be altered
upon conditioning with quinine; (2) to test if sleep deprivation can impair the learning capacities
and STM retention of D. melanogaster, and (3) to compare learning capacities and STM retention
among non-sleep-deprived and sleep-deprived D. melanogaster with supplementation.
Significance of the Study
For more than a century and particularly in the last 40 years (Vosshall, 2007), behavioral
assays designed for D. melanogaster have been successful in elucidating facets of the basis of
learning and memory in this model organism. This study contributes in these efforts by evaluating
and expanding the potential of APS assay in neurobehavioral research, particularly in assessing
pharmacological effects on Drosophila learning and STM using this simple assay format.
Conclusive confirmation of the experimental reliability of this tool will also strengthen support for
mainstreaming the use of APS assay in fly learning and memory studies. This would have positive
economic and practical implications in Drosophila learning and memory research worldwide.
The behavioral performance of D. melanogaster individuals in the assay can also provide
information on the effects of B. monnieri supplementation on non-sleep-deprived and sleep-
deprived Drosophila. Future efforts on the animal model may then relate the results obtained to
better evaluate the effects of these nootropics among humans in conditions like jet lag, sleep
deprivation, and night shift-requiring jobs.
2
Scope and Limitations of the Study
The study was limited to the assessment of learning and STM retention capacities in seven-
day old adult wild-type female D. melanogaster individuals, with disrupted sleeping patterns that
are fed with the different concentrations of B. monnieri supplement, based on their collective
average pass rates in the APS assay. Neither biochemical nor physiological mechanism as basis
for the results were experimentally elucidated in this study, and such processes were only
predicted, inferred and related to the collected data through literature.
3
REVIEW OF RELATED LITERATURE
Behavioral Learning Assays
Positive phototaxis is one of the earliest neurobiological aspects which was studied
extensively in Drosophila. As interest in genetics has been the main historical driving force in fruit
fly research, initial inquiries into this feature all attempted to link this apparently plastic behavior
with the inherited genotype and the surrounding environment (Hadler, 1964b). Hadler (1964a),
Hirsch and Boudreau (1958) and Médioni (1963) demonstrated that Drosophila phototaxis can be
altered when subjected to experimental behavioral-genetic methods. Such procedures include
quantitative screening, mathematical analyses of heritability and selective breeding performed
over several fly generations. Combining these techniques, Benzer (1967) created one of the first
Drosophila behavioral assays. In his first foray into this research field, he devised a method that
was able to “fractionate” D. melanogaster populations fed with the mutagen ethyl methane
sulfonate based on their relative phototaxic preferences on repeated trials. This approach facilitated
the isolation of non-phototaxic Drosophila behavioral mutants in just one offspring generation. As
this discovery effectively posited that behavior is genetically-controlled, the official acceptance of
the science of neurogenetics followed suit (Vosshall, 2007).
Research attention in phototaxis, however, gradually faded in favor of olfactory-based
memory, which was perceived to have greater potential in understanding neurobehavior in this
organism. Two landmark procedures concerned in the study of olfactory memory, by Quinn et al.
(1974) and Tully and Quinn (1985), are among the most replicated in Drosophila learning and
memory for more than 30 years. Olfactory-based learning in most insects, including Drosophila,
can be traced to structures in the nervous system called mushroom bodies. It is a cluster of about
2500 neurons known as Kenyon cells. The elucidation of the critical role of mushroom bodies in
4
olfactory-based memory involved a series of experiments: chemical ablation, induced genetic
mutation and radiofrequency ablation (de Belle and Heisenberg, 1994).
It was long assumed that the mushroom bodies has the same level of importance in other
observable behaviors in Drosophila (Waddell and Quinn, 2001). However, Wolf et al. (1998)
demonstrated that the ablation of the organ did not hamper the performance of the flies in simple
motor, tactile and visual tasks. Supporting this report in visual-based learning is the observation
that the presence of input from the optic lobes to mushroom bodies is non-existent (Heisenberg,
2003). According to van Swinderen (2011), visual-based learning studies in Drosophila has a
functional significance in higher and more complex tasks. This finding implies that visual
information reaches mushroom bodies and that genetic or chemical deactivation of this structure
still allows stimuli sensing and response and learning acquisition. However, visual information
hampers the ability to form associations and retrieve memories to the subtraction of unreliable
background cues from relevant ones to use the learned behavior in a partially different set of
circumstances (Waddell and Quinn, 2001). This organ complex is also shown to be used in
resolving contradictory visual cues. In mushroom bodies-disrupted flies, for instance, preference
of certain cues is attributed to pertinent cues instead of learning (Heisenberg, 2003; Liu et al.,
1999; van Swinderen, 2011). Modulation of selective fixation behavior based on the strength of
cues is associated to this structure as well (Xi et al., 2008). Brembs and Wiener (2006) suggests
that the use of mushroom bodies in visual-based memory is only for the simple stimulus processing
and not for cognitive and higher-order learning. Based on these uncertainties, molecular and neural
underpinnings of learning and memory through vision in Drosophila are arguably and relatively
less known than those through olfaction (van Swinderen, 2011).
5
While olfactory-based behavior of Drosophila is continually expounded through the
knowledge of mushroom bodies, the physiological basis of visual-based behavior is poorly
understood. Nevertheless, a wide range of assays have been designed for investigating this feature
in Drosophila. One of the earliest visual-based learning protocols was devised by Quinn et al.
(1974). It was utilized in many studies that followed. It consists of an operant group conditioning
apparatus forking into two arms: one transmitted light at a wavelength of 450 nm (blue) and the
other at 610 nm (red). Flies were conditioned to avoid the arm and the light that contained a bitter-
tasting compound known as quinine. Ever since this successful conditioning, quinine has been
used in several experiments on neurobiological behaviors of Drosophila. Quinine is used as a
potential stimulus for conditioning since it does not lead to stimulus sensitization or habituation. \
In aversive phototaxic suppression (APS) assay, an individual fly is operantly conditioned
to avoid light by associating it with quinine. This conditioning occurs when the fly exits the maze
through the lighted tube, where it encounters a quinine-soaked filter paper lining. The tendency to
avoid the lighted tube and to prefer the dark vial instead increases significantly over repeated
training (Le Bourg and Buecher, 2002; Le Bourg, 2005; Perisse et al., 2007; Seugnet et al., 2008;
Seugnet et al., 2009).
Effects of Sleep Deprivation on Behavior
The influence of sleep deprivation is well studied in Drosophila. In the review of sleep
deprivation-related research, Seugnet et al. (2008) showed that sleep loss impairs learning and
short sleep mutants impairs memory. Moreover, waking experience affects sleep needs in
Drosophila (Ganguly-Fitzgerald, 2006). All these studies show that sufficient sleep is important
in learning and memory.
6
Resting Drosophila is defined as a long period of immobility and an increased arousal
threshold at a particular time during the circadian day (Hendricks et al., 2000; Shaw et al., 2000).
Young flies tend to sleep extensively than older flies. Thus, sleep in the latter is modulated by
hypnotics and stimulants. The brain activity of Drosophila is reliably correlated with activity state,
and local field potential fluctuations can be reliably recorded from the brains of awake, moving
fruit flies (Nitz et al., 2002).
Xinjian et al. (2009) experimented on sleep deprivation and found that sleep deprivation
specifically impaired one-hour memory in wild type Canton-S flies. Flies were also tested on
alternative stresses like heat stress, oxidative stress, starvation and rotation stress and resulted in a
similar effect, leaving the memory of the flies intact. Mechanistic studies demonstrated that flies
with either silenced transmission of the MB during sleep deprivation or down-regulated cAMP
levels in the mushroom body demonstrated no sleep deprivation-induced one hour memory
impairment (Xinjian et al., 2009). In line with the expansion of knowledge and studies about the
effects of sleep deprivation on learning and memory, an emerging class of supplements called
nootropics are designed to enhance cognitive function (Dimond and Brouwers, 1976).
Nootropic Effects
Nootropics are part of a broader category of drugs known as performance and image
enhancing drugs which are used for enhancement of memory and cognition, sexual performance,
athletic performance or musculature (Stäubli et al., 1994). Dimond and Brouwers (1976) reported
that nootropics improved verbal learning, short-term memory, and performance in normal adults.
Nootropics are chemically similar to gamma-aminobutyric acid (GABA) which is the major
excitatory neurotransmitter in the brain. It has been suggested that these supplements act on the
7
cerebral cortex and related telencephalic structures and thereby enhance higher cortical functions
without apparent stimulant effects (Giurgea, 1973; Union Chimique Beege, 1976; Simeon et al.,
1980). Among other natural nootropics which claims to enhance learning and memory retention
is the plant Bacopa monnieri.
The plant Bacopa monnieri, also referred to as Herpestis monniera, is known to be
powerful antioxidants, and may also have anti-inflammatory properties (Jain et al., 1994; Tripathi
et al., 1996; Bhattacharya et al., 2000). Compounds responsible for the pharmacological effects of
B. monnieri include alkaloids, saponins and sterols. In the 1950s and 60s research by chemists at
the Central Drug Research Institute (CDRI) of India identified a variety of saponins. The main
active chemical constituents of Bacopa are the dammarane-type triterpenoid saponins (Garai et al.,
1996a, 1996b; Mahato et al., 2000) with jujubogenin and pseudojujubogenin as the aglycones
(Deepak and Amit, 2004). The saponins consist of numerous subtypes designated as bacosides,
bacopasides and bacopasaponins. Detailed investigations first reported the isolation of the alkaloid
“brahmine” from B. monnieri (Bose and Bose, 1931). In 1963, two active memory-enhancing
molecules were isolated and identified as Bacosides A and B.
Morgan and Bone (1999) stated that in herbal extracts standardised to bacoside A content,
it is likely that bacoside A collectively refers to the overall content of dammarane saponins rather
than a solitary saponin. Dammarane-type saponins have been shown to promote memory
acquisition, retention and retrieval in rats (Ma and Yu, 1993; Ma, Yu and Chen, 1991) and survival
of experimentally damaged neurones in chicken and rat cerebral cortex (Himi et al., 1989).
Commercially prepared nootropics that contains B. monnieri were developed to improve
learning and provide a feeling of well-being (Singh and Dhawan, 1982). Learning ability in rats
8
has been significantly enhanced by Bacopa extract as it facilitated acquisition, consolidation and
retention of three newly learned behavioural responses at an oral dosage of 40 milligram for three
times daily (Singh and Dhawan, 1982). Additionally, this study showed that the bacosides caused
enhanced levels of protein kinase activity and increases in protein levels in the hippocampus,
cerebral cortex and hypothalamus regions of the brain. The findings indicated positive implications
for improved neurotransmission and repair of damaged neurons via enhanced regeneration of
nerve synapses.
Placebo-controlled double-blind phase II clinical trials were held by the CDRI on children
with attention deficit hyperactivity disorder (Pal and Sarin, 1992). Additional studies were done
for the elderly with age-associated memory impairment. Both tests showed that B. monnieri
supplement is safe to use. At the Swinburne University in Australia, double-blind randomized
placebo-controlled studies were conducted. Tests results showed that Brahmi significantly
improved visual information processing (Shanker and Singh, 2000).
9
MATERIALS AND METHODS
Test Animals
Wild type Drosophila melanogaster (Oregon-R strain) were obtained from the Department
of Biology, University of the Philippines in Baguio. The flies were reared based on the protocol
by Medina et al. (2015) at room temperature on a sweet potato medium. A bottle containing one-
day old flies from hatched pupa were immediately transferred to two separate bottles containing
freshly prepared sweet potato medium. The reproduction of flies were allowed to obtain sufficient
number of flies. The breeding bottles were maintained at 24°C to 28°C and in a 12-hour light: 12-
hour dark cycle, with the 12-W fluorescent light. The progeny of these flies was considered for for
the test group of this study. However, only female flies were used since male flies spend
considerable time sleeping during both day and night, with the amount of sleep during the day
often comparable to that at night. Although mature females sleep as much as their male
counterparts during the night, they exhibit sustained activity during day and sleep much less during
this period (Huber et al., 2004). Thus, females were seen to be more consistent models for the
study (Dalke, 2002).
Carbon dioxide gas was used to put the flies to sleep while segregation according to sex
was conducted. Sex combs were used to distinguish male from the female flies since the structure
is present in male Drosophila only.
To ascertain that all flies that were used in the experiment are seven days old, separating
the progeny in a stock bottle with respect to the day of juvenile emergence was necessary. If, for
the first time, the newly-emergent flies were observed in a stock bottle in the morning (0700 hrs),
transfer to a new bottle, with medium corresponding to that in their current bottle, was performed
10
before midnight (<2330 hrs). All transferred flies were then considered as one-day old adults the
next day.
Test Chemicals
The Bacopa monnieri supplement, with brand name Memo Plus Gold, that was used, the
range of dosages employed, and the proposed mechanism of action were summarized in Appendix
1b. Flies were raised in the media containing a specific dose of the supplement (g/mL) at 25°C.
The supplement was only added into the media at 60°C before it solidifies. A lethality test was
conducted to estimate what concentrations are sublethal and effective to produce measurable
effects on the learning capacities and STM retention of D. melanogaster. The baseline dosages of
the supplements for Drosophila were calculated using the equation by Hong et al. (2002) for
conversion from human dosages. A five-point series of B. monnieri concentrations starting from
4.62 in g/mL and decreased by ten-fold reaching 4.62 x 10-4 g/mL were used for the lethality test.
Three concentrations were chosen for the experiment proper and were labeled as low, mid and
high, respectively. Female flies were allowed to feed on the food medium with corresponding B.
monnieri concentration previously defined from the first day after eclosion till the seventh day
when learning and conditioning commenced. There was constant feeding of the supplement during
the sleep deprivation process and resting period.
To prepare the quinine solution used as an aversive stimuli in the “lighted” tube, 1.98 grams
of quinine hydrochloride was dissolved in 50 mL distilled water to prepare a 0.10 M stock solution.
Sleep Deprivation Procedure
11
Flies were sleep-deprived using an improvised Sleep Nullifying Apparatus (SNAP)
adapted from Shaw et al. (2002). They were individually put inside test tube-containing-media.
The SNAP travels vertically such that sleeping flies were displaced during the dropping movement
of the machine once a distance of five centimeters was reached for 15 times every minute. The
whole sleep deprivation process lasted for ten hours. Flies were sleep-deprived using the SNAP
from zeitgeber time ZT 12 to ZT 22. Flies were confirmed to be kept awake by their constant
moving inside the tubes.
Pre-tests
1. Quinine Sensitivity
This pre-test was conducted using the T-maze. It consisted of a center column with the trap
door and two independent chambers, a dark chamber and a lighted chamber. Two 15 mL plastic
centrifuge tubes were used to prepare it. One tube was shone with light to serve as the lighted
chamber. The other tube was wrapped with aluminum foil to serve as the dark chamber. A 180 μL
of either distilled water or quinine solution were added to filter paper and placed in the lighted
chamber, two centimeters from the opening. These two chambers were screwed on each side of
the center column of the T-maze with the trap door in the middle closed (Appendix 2).
Sensitivity to quinine was evaluated as in Le Bourg and Buecher (2002) with the following
modifications: 30 flies were individually placed in the dark tube of the T-maze that was maintained
in a horizontal position after the introduction of the animal. Each half of the apparatus contained
separate pieces of filter paper that could be wetted with quinine or kept dry. The quinine sensitivity
index (QSI) was determined by calculating the time in seconds that the fly spent on the dry side of
the tube when the other side had been wetted with quinine, during a five minute period. In the
12
absence of quinine when both sides of the tube are dry, flies quickly move to the side opposite
their point of entry and stay there. Indeed, under dry conditions, flies never reside for more than
one minute on the side of the chamber where they were placed initially (Le Bourg and Buecher,
2002). However, in the presence of quinine, flies briefly cross from the dry end of the tube into
the quinine portion of the apparatus and then quickly come back to the dry side of the chamber (Le
Bourg and Buecher, 2002).
2. Velocity Stress Test
Seven-day old flies experienced jolting using the SNAP, 30 times per minute for ten hours
from ZT 12 to ZT 22, and then were given 30 minutes to recover before being subjected to the
APS assay.
Average Pass Rates
Incidence of flies from originally the dark chamber that goes through the lighted chamber
was recorded and tallied per trial except during photopositive screening. A score of “1” was given
for a fly exhibiting a photonegative choice (remained in the dark chamber). A score of “0” was
noted for a fly entering the lighted chamber. The trials that consecutively scored with 1 up to the
last four trials were summed and divided by total number of trials to get the pass rate. The average
pass rate (APR) or the mean of all the pass rates of the 30 flies per treatment group was then
obtained together with its standard deviation and error. These values were then used to represent
the percent learning and memory retention in the descriptive statistical methods that were
performed.
13
Assay Proper
The aversive phototaxic suppression assay (LeBourg et al., 2002) takes advantage of the
positive phototactic behavior of flies to train them to associate light with an aversive odor, in this
case quinine.
The following were the treatment groups used in the experiment: (1) non-sleep-deprived
and fed without supplement or slp+/Bm-, (2) non-sleep-deprived and fed with the supplement with
three sub-setups or slp+/Bm+ low, slp+/Bm+ mid and slp+/Bm+ high, (3) sleep-deprived and fed
without the supplement or slp-/Bm-, and (4) sleep-deprived and fed with the supplement with three
sub-setups or slp-/Bm+ low, slp-/Bm+ mid and slp-/Bm+ high. A sample size of 30 flies were tested
per setup/sub-setup.
During the experimental procedure, flies were individually screened for positive
phototaxis, subjected to SNAP (depending on assigned treatment group), trained to suppress their
preference for light and then tested for the retention of the conditioned behavior. They underwent,
a general sequence of steps comprising an experimental trial which is the basic unit of the assay
procedure.
1. Photopositive Fly Screening
The dark chamber was unscrewed from the T-maze to allow transfer of a fly into it then
screwed back to the T-maze immediately after. The lights in the room were turned off and the red
light was turned on. Acclimatization of the flies in the dark chamber were allowed for 30 seconds
before slowly turning on the light source illuminating the lighted chamber. The trap door
separating the two chambers were then slowly opened. If the fly walked towards the lighted
chamber within ten seconds, it was considered as positively phototactic and qualified for the assay.
14
This single-trial phase was conducted to qualitatively verify the presence of the positive phototoxic
reflex in the fly. If the fly did not show this behavior, it was not considered as photopositive
therefore, not used in the experiment since APS conditioning relies on the photopositive trait to
induce learning in the first place. Furthermore, as this behavior is essentially a behavioral reflex,
only one screening trial was performed. Doing this trial more than once might inadvertently alter
tube preference in the succeeding blocks and irrespective of the effect of the conditioning
procedure.
Fruit flies are known to be generally photopositive, hence they would prefer most of the
time to travel to the lighted tube. However, it is also observed that variation in the level of
preference to light exists even among wild-type fly groups, from highly photopositive to
practically photonegative (Hadler, 1964a; Benzer, 1967; Markow, 1975).
2. Learning Phase
For the APS assay proper, a fly that demonstrated positive phototaxis was tapped back to
the dark chamber. The trap door was closed and the lights were turned off. Thirty seconds were
allowed for acclimatization but during this time, a filter paper with quinine solution was put inside
the lighted chamber. Then, the trap door was slowly opened as well as the lights. The fly was given
10 seconds to walk on the quinine coated lighted chamber. This was repeated for nine times.
Usually, wild type flies avoid after three to five training trials (Ali et al., 2011). Failure to walk to
the lighted chamber was recorded as "Pass", which is equivalent to "task learned through
reinforcement".
15
The right-left (RL) double alternation of the tubes was executed in an RRLLRRLLRR
manner, switching every two trials. This alternation was performed to exclude sustained
directional bias as a source of the behavioral response (Le Bourg and Buecher, 2002).
3. Short-Term Memory Retention Phase
To assess STM retention in flies, each fly was placed back into its original food tube and
kept aside for three hours. Nootropic feeding was resumed during this resting period. Three hours
post training, each fly was put in the T-maze again in the same way as before, and the number of
times the fly avoids (pass) or goes into (fail) the lighted chamber was recorded. A total of five
trials were conducted to test STM retention. Alternation of the tubes was performed after every
trial. This phase was conducted to assess the span of effective retention, handling and retrieval of
the memory of the conditioned behavior in the flies during the one-hour STM period (Ramaswami,
2003; Tamura et al., 2003).
Statistical Analysis
To detect if differences in obtained average pass rates were significant due to Bacopa
monnieri supplement, sleep deprivation and the interaction of the two, a two-way analysis of
variance (ANOVA) was utilized. In addition, pairwise comparisons for each treatment group were
performed using Bonfferoni method. The level of significance (α) was set at 0.05.
16
RESULTS
Lethality Test
The group with no Bacopa monnieri supplementation had a 100% survival rate of flies.
The three lowest among the five tested concentrations showed the highest survival rate next to the
no supplement treatment group. Although notable mortality rates were still observable within one
week. The lowest B. monnieri concentration had a survival rate of 96.60% of the fruit flies. The
mid-level concentration, which is ten-fold higher, had a 90% survival rate. Lastly, 86.67% of the
fruit flies survived in the treatment group with the highest concentration of the supplement. The
last two setups with the concentrations 0.462 g/mL and 4.62 g/mL, had a survival rate of 60.00%
and 53.33%, respectively. It should also be noted that pupae formation from the two highest
concentrations were approximately 50% less compared to the three lowest concentrations
suggesting that at higher doses may have negative effects on fertility.
Quinine Sensitivity
There was no significant difference on the quinine sensitivity indices (QSI) between non-
sleep-deprived and sleep-deprived fruit flies (p<0.05). All flies in both treatment groups displayed
QSI scores well within ranges that permit normal learning together. Data were recorded alongside
17
the standard deviation (SD). Indeed, under dry conditions with filter paper only, flies did not reside
for more than one minute on the side of the chamber where they were placed initially (Le Bourg
and Buecher, 2002). However, in the presence of quinine, all flies in both treatment groups briefly
cross from the dark tube to the quinine on the lighted chamber of the apparatus and then quickly
go back to the dry side of the chamber.
Velocity Stress Test
The average pass rate of the fruit flies that had undergone the stress test were similar with
the APR values of the slp-/Bm- treatment group. Two-way analysis of variance (ANOVA) results
indicated that the increased rotations per minute of the sleep deprivation machine had no
significant difference on the learning and memory retention capacity of the fruit flies in comparison
with the standard rotations per minute.
Learning Capacity
The APR values of the slp-/Bm- treatment group was 45.92%. APR values of all sleep-
deprived treatment groups decreased in comparison with the APR values of non-sleep-deprived
groups. All treatment groups with B. monnieri supplementation had increased APR values in
comparison to the no supplementation group (p<0.001). The slp+/Bm+ mid treatment group
obtained the highest APR of 81.48% among all treatment groups. The APR values of the slp+/Bm+
high group was 80.37%. The APR values of the two highest concentrations were higher compared
to that of the lowest (p<0.05). There was no significant difference between the APR values of the
two highest concentrations (p>0.05). The APR values obtained in the slp-/Bm+ groups were lower
18
compared to the APR values of the slp+/Bm+ groups (p>0.05). There was no interaction between
these two factors that may affect the resulting rates (p<0.01).
Short-term Memory Retention
After three hours post-training, the APR values of the slp-/Bm- treatment group was
47.33%. APR values of all sleep-deprived treatment groups decreased in comparison with those
of not sleep-deprived groups. There was an increase in the APR values of the groups slp+/Bm+
low, slp+/Bm+ mid and slp+/Bm+ high in comparison to slp+/Bm- (p<0.001). Similarly, the APR
values of the slp-/Bm+ group were higher in comparison to that of slp-/Bm- (p<0.001). The STM
retention rates of the two highest concentrations were higher compared to that of the lowest
(p<0.001). There was no significant difference between the APR of the two highest concentrations
(p>0.05). The APR values of the sleep-deprived flies with the mid and low concentrations were
not significantly different with the non-sleep-deprived counterparts (p>0.05). There was a decrease
in the APR values of the group slp-/Bm+ low in comparison to its non-sleep-deprived counterpart.
Interaction between two factors had non-existent effect and insignificant to the obtained short-term
memory retention rates (p>0.05).
19
DISCUSSION
As an inexpensive and simple assay, aversive phototaxis suppression (APS) assay may be
utilized to evaluate learning, short-term memory (STM) retention and response inhibition in
relation to genetic and environmental manipulations (Seugnet et al., 2009; van Swinderen, 2011).
With the exclusion of the positive phototaxis screening trial and the quinine sensitivity index (QSI)
test, the assay procedure is divided into two sequential phases: the learning phase and the STM
retention phase. Removing quinine during the APS assay also removes a contextual cue, humidity,
which is required for recall. Thus, in the STM retention phase, filter paper soaked in water was
used as a recall cue for the flies.
The presence study utilized quinine as the aversive stimulus for conditioning the avoidance
of light by D. melanogaster. Quinine sensitivity was tested in order to assess its induction of
avoidance behavior in both non-sleep-deprived and sleep-deprived flies. It was observed that when
quinine was used in the T-maze test, flies briefly cross from the dark chamber of the tube into the
quinine portion of the lighted chamber and then quickly come back to the dry side of the chamber.
Moreover, quinine sensitivity of flies did not differ in non-sleep-deprived and sleep-deprived
20
conditions suggesting that contact chemosensation of the bitter tasting compound is not
significantly altered by sleep deprivation.
Since then, sleep deprivation experiments have been inherently problematic in that it is
frequently difficult to test whether an observed outcome is because of the lack of sleep or the
methods used to keep the organism awake (Rechtschaffen and Bergmann, 2002). (Insert studies).
Statistically, the APR values of learning and STM retention of the control and the one subjected
to velocity stress were similar indicating that the reduction of learning capacity and STM retention
is specifically imparted by the sleep deprivation alone. In other fly experiments concerning
neurobiological a study by Shaw et al. (2002) have already demonstrated that the SNAP used to
keep animals awake does not activate stress-response genes such as those involved in metabolic
stress, chemical stress, and humoral stress.
From the data gathered, a ten hour sleep deprivation decreases both the learning ability and
the STM retention of the flies. Low motivation is an unlikely explanation for the impairment of
learning since the number of trials it takes for a specific fly to learn was statistically lower from
the non-sleep-deprived flies that were not fed with B. monnieri supplement. Sleep deprivation also
did not alter the QSI which indicates that learning impairment is due to sleep deprivation and not
due to sleep deprivation-induced alteration in sensory thresholds. The decline of STM retention
in sleep-deprived flies may be attributed to the deficit in mushroom body plasticity (Seugnet et
al., 2008) during episodic memory encoding, resulting in impairment of subsequent retention
(Talamini et al., 2008).
Learning was observed in all non-sleep-deprived and sleep-deprived D. melanogaster as
confirmed by the consistent avoidance of a fly from the light in the last four trials of the learning
phase. This indicates that effective conditioning was qualitatively observed. The APR values
21
recorded for the non-sleep-deprived flies fed with the different B. monnieri supplement
concentrations were statistically similar to the sleep-deprived flies fed with the same B. monnieri
concentrations. In fact, even at low concentrations used in the study, B. monnieri supplement
increases learning in sleep-deprived flies at the same level as their non-sleep-deprived
counterparts. The results demonstrate that B. monnieri supplement compensates for deficiency of
learning caused by sleep deprivation. Comparisons of the three concentrations of the B. monnieri
supplement on both non-sleep-deprived and sleep-deprived flies occurred at a dose–dependent
manner, reaching a statistical plateau starting on the middle dosage. Moreover, the treatment
groups with B. monnieri supplement had a faster rate in the first phase, learning as early as Trial 2
or 3, indicative of better learning capacities, than the one without B. monnieri supplement.
The results of the STM retention phase showed that B. monnieri supplement also enhances
neural processes involving memory retention in a dose-dependent manner. For both non-sleep-
deprived and sleep-deprived flies, significant increase of STM retention was observed in the two
highest concentrations. No additional functional and temporal benefit on the neural processes for
STM retention will be observed after the middle dosage has been reached. In this study, a threshold
concentration for the positive pharmacological impact of the compound for STM retention exists,
which is between lowest dosage and middle dosage. A statistical difference can be seen between
the non-sleep-deprived and the sleep-deprived flies that was fed with the lowest dosage of B.
monnieri. This finding suggests that if little experiential information was formed in the first place
during learning phase of the sleep-deprived flies, then the recent learned behavior that can be
retained and retrieved for use in future encounters with the same trial conditions would be much
smaller. The effect of B. monnieri supplement on the differential and increasing STM retention
trends were likely manifestations of (1) enhancement of B. monnieri supplement on memory phase
22
that following a dose dependent manner, and (2) the natural events outlining the extinction of the
memory of the conditioned behavior. In the context of the APS assay, the more frequently a trained
fly avoids the light, the more likely the behavior will be retained. In fact, it parallels to the cellular
basis of learning and memory retention (McGuire et al., 2005). This retention is an indicator of the
potentiation of the STM. The persistent strengthening of synapses of the test flies will lead to long-
term potentiation (LTP). Memory formation is dependent on changes in synaptic efficiency that
permit strengthening of associations between neurons (Heisenberg, 2003).
A plausible mechanism of mode action of this nootropic relates the effect of the bacosides
in neuron by increasing levels of serotonin when present in the nervous system in corresponding
rate of Ca2+ influx during the ionotropic pathway of LTP (Singh and Dhawan, 1997). The increase
of serotonin levels saturate the binding sites of serotonin receptors and allows the passage of more
Ca2+ ions into the mushroom body which activates protein kinase C and calmodulin kinase, causing
substrate level phosphorylation and insertion of additional serotonin receptors (McGuire et al.,
2005). The addition of more serotonin receptors to the membrane causes the post-synaptic cell to
respond more strongly to future releases of serotonin to a certain extent until all receptors are
saturated with serotonin. The synapse is thus strengthened and potentiation has occurred.
Strengthening of these synapses due to nootropic agents are demonstrable in changes of
insect behavior. In the context of fruit fly lifestyle, plant-derived nootropic compounds obtained
through feeding can have an effect on memory requiring tasks for these insects such as courtship
behavior in the wild. For example, male Drosophila show a suppression in courtship that can last
for three hours after encountering a mated female (Ackerman and Siegel, 1982). If such STM
retention observed in the study were to occur in the males through nootropic consumption, memory
of such vital details will be enhanced, decreasing the chance that the male fly to again attempt
23
courting the same females that avoided him. This event will lead to a conservation of energy and
an increase in reproductive success over time (Siegel and Hall, 1979; Griffith and Ejima, 2009).
The implication of the inclusion of natural nootropic in a daily-based diet for other species
may improve mental performance, without the risks of side effects. Wide ranges of natural
nootropics range from artichoke leaves to the Chinese club moss plant and even amino acids like
tryptophan which is found in poultry products and theanine that naturally occurs in green tea can
be included in a daily dietary plan. In dietary science, proper combination of natural nootropics,
known as nootropic stacking, synergistically enhances the efficacy of each active ingredient of the
nootropic (Spencer, 2009). Hence, having a diet which includes a natural nootropic may be able
to deter failures of mental performances including sleep deprivation.
These observations imply that the enhancing effects of B. monnieri supplement on learning
and STM retention in D. melanogaster follows a dose-dependent manner, with the optimal level
between the lowest and the middle dosage. In addition, having a natural nootropic diet may be able
to deter failures of mental performances including sleep deprivation.
24
CONCLUSION
The effects of Bacopa monnieri supplementation enhances both the learning capacities and
short-term memory retention of both non-sleep-deprived and sleep-deprived seven-day old female
fruit flies. In addition, advantageous effects were dose-dependent from the low, 4.62x10-4g/mL to
the mid, 4.62x10-3g/mL concentrations for both learning and short-term memory retention with
middle dosage being the optimal concentration among the three concentrations used.
25
RECOMMENDATIONS
The independence of the nootropic on the sleep-deprived condition signifies possible
similar effects to other neurobiological conditions. Future efforts are hence suggested to more
accurately test the different neurobiological conditions on humans with measurable doses in its
optimum condition. Comprehensive studies following suit may soon be able to use the information
from Drosophila research in identifying the biologically-equivalent doses for learning and memory
retention in humans.
To test whether the observed behavior to alterations in certain biological structures are due
to the effects of the nootropic, biochemical and anatomical screenings of nervous tissue regions
are also recommended. These techniques may prove useful in monitoring developmental stage-
and age-specific effects of B. monnieri supplement intake. Furthermore, such approach can be of
help in investigating the impact of the nootropic in natural Drosophila activities such as courtship
and foraging.
The APS assay is also recommended to be used in conjuction with pharmacological effects
(i.e. other drugs and compounds) on learning and STM retention. This can be done by evaluating
the impact of other compounds believed or known to alter neurophysiological and behavioral
capacities, such as alcohol and caffeine. Finally, further attempts to improve the APS protocol
developed and tested here are urged to be conducted, since the increase in the quality and relevance
of the obtained data are dependent on such efforts.
26
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33
TABLES
Table 1. Difference on the Quinines Sensitivity Index (QSI) of both non-sleep-deprived and sleep-
deprived D. melanogaster. All flies display QSI scores well within ranges that permit
normal learning. The final QSI is the average of the scores obtained for 30 flies ± standard
deviation (SD) values.
Fly Type ( n=30 ) Quinine Sensitivity Index
Non-Sleep-Deprived D. melanogaster 4:29 ± 0:13
Sleep-deprived D. melanogaster 4.34 ± 0:18
34
Table 2. Average Pass Rates for Learning and Memory Retention of non-sleep-deprived
Drosophila
Fly Bm- Bm- Bm+ low Bm+ low Bm+mid Bm+ mid Bm+ high Bm+ high
number Learning MR Learning MR Learning MR Learning MR
1 44.44% 60.00% 77.78% 80.00% 77.78% 100.00% 88.89% 80.00%
2 66.67% 80.00% 66.67% 60.00% 88.89% 80.00% 88.89% 80.00%
3 44.44% 80.00% 77.78% 80.00% 88.89% 80.00% 77.78% 60.00%
4 77.78% 80.00% 88.89% 60.00% 77.78% 80.00% 88.89% 80.00%
5 44.44% 80.00% 66.67% 80.00% 88.89% 80.00% 66.67% 100.00%
6 44.44% 80.00% 66.67% 60.00% 88.89% 100.00% 66.67% 100.00%
7 44.44% 80.00% 88.89% 80.00% 55.56% 100.00% 88.89% 80.00%
8 66.67% 60.00% 55.56% 60.00% 88.89% 60.00% 55.56% 80.00%
9 77.78% 80.00% 55.56% 60.00% 88.89% 100.00% 88.89% 80.00%
10 55.56% 60.00% 77.78% 80.00% 88.89% 80.00% 88.89% 60.00%
11 66.67% 80.00% 66.67% 80.00% 66.67% 60.00% 66.67% 60.00%
12 66.67% 80.00% 88.89% 80.00% 77.78% 80.00% 88.89% 80.00%
13 66.67% 80.00% 66.67% 80.00% 77.78% 100.00% 77.78% 100.00%
14 55.56% 60.00% 77.78% 80.00% 88.89% 100.00% 88.89% 60.00%
15 66.67% 40.00% 66.67% 60.00% 88.89% 80.00% 77.78% 100.00%
16 55.56% 60.00% 77.78% 60.00% 88.89% 100.00% 88.89% 60.00%
17 66.67% 40.00% 88.89% 60.00% 77.78% 60.00% 88.89% 80.00%
18 66.67% 40.00% 66.67% 80.00% 88.89% 40.00% 88.89% 100.00%
19 55.56% 60.00% 55.56% 60.00% 66.67% 100.00% 55.56% 60.00%
20 44.44% 60.00% 44.44% 60.00% 88.89% 40.00% 77.78% 80.00%
21 66.67% 40.00% 77.78% 80.00% 88.89% 80.00% 77.78% 100.00%
22 77.78% 40.00% 88.89% 80.00% 66.67% 100.00% 55.56% 80.00%
23 44.44% 40.00% 66.67% 80.00% 55.56% 80.00% 66.67% 100.00%
24 66.67% 40.00% 66.67% 80.00% 88.89% 40.00% 88.89% 80.00%
25 55.56% 40.00% 66.67% 80.00% 88.89% 80.00% 88.89% 60.00%
26 55.56% 60.00% 66.67% 40.00% 88.89% 60.00% 77.78% 60.00%
27 66.67% 60.00% 77.78% 60.00% 88.89% 80.00% 88.89% 60.00%
28 55.56% 40.00% 88.89% 60.00% 66.67% 100.00% 88.89% 100.00%
29 44.44% 60.00% 77.78% 40.00% 88.89% 100.00% 88.89% 60.00%
30 88.89% 40.00% 77.78% 60.00% 77.78% 80.00% 88.89% 100.00%
Average
60.00% 60.00% 72.60% 68.67% 81.48% 80.67% 80.37% 79.33%
Pass Rate
35
Table 3. Average Pass Rates for Learning and Memory Retention of sleep-deprived Drosophila
Fly Bm- Bm- Bm+ low Bm+ low Bm+mid Bm+ mid Bm+ high Bm+ high
number Learning MR Learning MR Learning MR Learning MR
1 55.56% 20.00% 77.78% 40.00% 88.89% 100.00% 88.89% 60.00%
2 66.67% 60.00% 66.67% 60.00% 77.78% 80.00% 55.56% 100.00%
3 55.56% 60.00% 88.89% 60.00% 77.78% 80.00% 66.67% 80.00%
4 44.44% 80.00% 66.67% 80.00% 77.78% 80.00% 88.89% 40.00%
5 44.44% 0.00% 66.67% 60.00% 66.67% 80.00% 66.67% 80.00%
6 44.44% 40.00% 66.67% 60.00% 66.67% 80.00% 77.78% 40.00%
7 55.56% 80.00% 88.89% 80.00% 66.67% 80.00% 55.56% 60.00%
8 66.67% 60.00% 77.78% 60.00% 55.56% 80.00% 88.89% 80.00%
9 44.44% 40.00% 77.78% 60.00% 88.89% 100.00% 66.67% 80.00%
10 44.44% 40.00% 55.56% 20.00% 66.67% 80.00% 88.89% 60.00%
11 44.44% 60.00% 88.89% 60.00% 77.78% 60.00% 66.67% 40.00%
12 55.56% 60.00% 88.89% 60.00% 88.89% 100.00% 88.89% 80.00%
13 44.44% 40.00% 66.67% 40.00% 88.89% 80.00% 88.89% 100.00%
14 44.44% 40.00% 66.67% 60.00% 66.67% 80.00% 66.67% 60.00%
15 0.00% 0.00% 66.67% 60.00% 88.89% 100.00% 77.78% 80.00%
16 44.44% 80.00% 66.67% 40.00% 66.67% 40.00% 66.67% 80.00%
17 44.44% 80.00% 55.56% 60.00% 88.89% 80.00% 88.89% 80.00%
18 44.44% 60.00% 77.78% 60.00% 88.89% 80.00% 88.89% 100.00%
19 55.56% 80.00% 77.78% 80.00% 77.78% 60.00% 88.89% 80.00%
20 44.44% 60.00% 44.44% 20.00% 66.67% 80.00% 77.78% 60.00%
21 44.44% 60.00% 44.44% 40.00% 88.89% 80.00% 77.78% 80.00%
22 44.44% 40.00% 66.67% 60.00% 88.89% 60.00% 88.89% 100.00%
23 44.44% 0.00% 66.67% 40.00% 66.67% 80.00% 66.67% 80.00%
24 44.44% 60.00% 88.89% 80.00% 66.67% 60.00% 66.67% 60.00%
25 44.44% 60.00% 55.56% 40.00% 88.89% 80.00% 88.89% 100.00%
26 66.67% 40.00% 77.78% 80.00% 66.67% 60.00% 77.78% 100.00%
27 0.00% 0.00% 77.78% 80.00% 66.67% 60.00% 66.67% 80.00%
28 44.44% 40.00% 66.67% 60.00% 88.89% 80.00% 66.67% 60.00%
29 44.44% 40.00% 77.78% 60.00% 88.89% 100.00% 88.89% 100.00%
30 55.56% 40.00% 77.78% 60.00% 88.89% 80.00% 55.56% 80.00%
Average
45.92% 47.33% 71.11% 57.33% 77.78% 78.00% 76.30% 76.00%
Pass Rate
36
FIGURES
Figure 1. Survival rate of Drosophila melanogaster on the difference of 10-fold concentration of

Bacopa monnieri Supplement over the course of seven days.
Figure 2. Comparison of the effect of velocity stress caused by mechanical stimulation of the
machine on the learning and short-term memory retention of D. melanogaster (n=30). The
results indicate that stress did not affect both learning and memory (P<0.05).
37
Standardized
Learning STM
Figure 3. Standardized average pass rates for learning and memory retention of non-sleep-
deprived and sleep deprived (slpD) D. melanogaster (n=30). Low dosage corresponds to 4.62 x
10-4 g/mL, middle dosage corresponds to 4.62 x 10-3 g/mL and high dosage corresponds to 4.62 x
10-2 g/mL. Error bars represent standard error (SE) values.
38
TRIALS TRIALS
1 2 3 4 5 6 7 8 9 1 2 3 4 5
TRIALS
1 2 3 4 5 6 7 8 9
slp+/Bm-
slp+/Bm+ low
slp+/Bm+ mid
slp+/Bm+ high
slp-/Bm-
slp-/Bm+ low
slp-/Bm+ mid
slp-/Bm+ high
PASS
FAIL
Figure 4. Pass-Fail Map for Learning (left) and STM Retention (right) of D. melanogaster
39
Figure 5. Mean score of photonegative choices (1 or 0) by non-sleep-deprived D. melanogaster

on the learning phase and short-term memory retention of different treatment groups
(n per group = 30)
Figure 6. Mean score of photonegative choices (1 or 0) by slpD D. melanogaster on the learning

phase and short-term memory retention of different treatment groups (n per group = 30)
40
APPENDICES
Appendix 1a. Media synthesis (adapted from the Medina et al., 2015).
1. Mix the following in a 2000-mL Erlenmeyer flask.
 Distilled water: 1000 mL
 Sweet Potato: 500 g
 Bacto ™ Agar: 15 g
 Baker’s yeast: 10 g
2. Boil the resulting mixture using autoclave.
3. Add 0.08% propionic when boiled media is cooled to 70°C.
4. Add yeast paste made with 0.08% propionic acid for optimum reproduction.
5. Place the content in the breeding bottles (approx. 450 mL per bottle).
6. Transfer the flies into the freshly prepared sweet potato medium.
Appendix 1b. Summary of the Bacopa monnieri Memory Enhancing Supplement
Memory-Enhancing Concentration That Will Be Proposed Mode of Action

Supplement Used (g/mL)
Improves consolidation of
learned behavior and delays
Bacopa monnieria 4.62 x 10-4 (Low) its extinction by enhancing
protein kinase activity and
4.62 x 10-3 (Mid)
new protein synthesis
4.62 x 10-2 (High) (Bhattacharya and Ghoshal,
1998).
a
Taking into account that 500 mg of capsulated B. monnieri supplement is equivalent to 55±5% w/w of
dried herb.
41
Appendix 2. T-Maze Setup for Aversive Phototaxis Suppression Assay. (A) The overall experimental
setup for the APSA experiment with the light source connected to the "lighted" falcon
tube, lined with filter paper and the "dark" tube on the left (covered in foil), separated by
a trap door. (b) During the training and testing phase, the fly is in the dark chamber and
the trap door opened after the light is turned on in the lighted chamber.
Trap Door
Light Chamber Dark Chamber
A.
Quinine Soaked
Filter Paper Red Light
Light Source
B.
42
Appendix 3. Sleep Nullifying Apparatus (SNAP) rhythmically jolts flies up and down, displacing
sleepy flies fifteen times per minute. In each trial, flies will be sleep deprived using
the SNAP from zeitgeber time (ZT) 0 to ZT 10 (beginning of the light phase).
Speed Controller
Unit
43
Appendix 4. Methodology flowchart. Flies were repeatedly introduced to the apparatus in

several trials.
TREATMENT GROUPS
Not Sleep-Deprived Group Sleep-Deprived Group
No No
Supplement w/ Bacopa monnieri w/ Bacopa manniera
Supplement
Supple Supple
Individual
fly
General Assay Procedure Pre-Tests

Velocity Quinine
Positive Phototaxis Screening Trial
Stress Test Sensitivity
immediately
Learning Phase
Aversive Phototaxic Suppression
3 h after
Memory Retention Phase
Per Treatment Group: Average Pass Rate Calculation
Statistical Analysis: Two-Way ANOVA, Bonferroni Method
44
APPENDIX 5
Analysis of Variance (ANOVA) and Post-hoc Analysis Results
Appendix 5a. Two way ANOVA results for Velocity Stress Test
Source of Variation % of total P-value P-value Significant

variation summary
Interaction 1725e-14 > 0.9999 Ns No
Row Factor 1.580 0.1750 Ns No
Column Factor 2.527e-7 0.9996 Ns No
SS df MS F P-value
Interaction 3.638e-12 1 3.638e-12 2.033e-14 P > 0.999
Row Factor 333.3 1 333.3 1.863 P = 0.1750
Column Factor 5.329e-5 1 5.329e-5 2.97e-7 P = 0.9996
Residual 20759 116 179
Appendix 5b. Two way ANOVA results for the Learning Phase
N=240 R-squared = 0.5054
Root MSE = 11.6144 Adj R-squared = 0.4905
Factor Partial SS df MS F Prob > F

Bacopa monnieri 28693.6687 3 9564.55625 70.90 0.0000
Sleep deprivation 1671.70103 1 1671.70103 12.39 0.0005
Bacopa + sleep deprivation 1615.48719 3 538.49573 3.99 0.0850
Appendix 5c. Pairwise Comparisons of Marginal Linear Predictions between the control and
treatment groups for the Learning Phase
Factor
Bacopa monnieri conc. Contrast Standard Error t P > |t|
low vs. none 20.373 2.12049 9.61 0.000
low vs. mid 27.59467 2.12049 13.01 0.000
low vs. high 25.55783 2.12049 12.05 0.000
mid vs. none 7.221567 2.12049 3.41 0.005
mid vs. high 5.184833 2.12049 2.45 0.091
high vs. none -2.036833 2.12049 -0.96 1.000
Sleep Deprivation
slpD vs. normal -5.278417 1.499413 -3.52 0.001
45
Appendix 5d. Pairwise Comparisons of Marginal Linear Predictions for the Learning Phase Using
Bonferroni Method
x1=B. monnieri treatment groups (0= none, 1= low, 2= mid, 3= high)

x2=sleep deprivation groups (0=normal, 1=slpD)
Treatment Groups (x1,x2) Contrast Std. Error t P > |t|

(0,1) vs. (0,0) -14.077 2.998826 -4.69 0.000
(1,0) vs. (0,0) 13.705 2.998826 4.57 0.000
(1,1) vs. (0,0) 12.964 2.998826 4.32 0.001
(2,0) vs. (0,0) 21.85233 2.998826 7.29 0.000
(2,1) vs. (0,0) 19.26 2.998826 6.42 0.000
(3,0) vs. (0,0) 20.371 2.998826 6.79 0.000
(3,1) vs. (0,0) 16.66767 2.998826 5.56 0.000
(1,0) vs. (0,1) 27.782 2.998826 9.26 0.000
(1,1) vs. (0,1) 27.041 2.998826 9.02 0.000
(2,0) vs. (0,1) 35.92933 2.998826 11.98 0.000
(2,1) vs. (0,1) 33.337 2.998826 11.12 0.000
(3,0) vs. (0,1) 34.448 2.998826 11.49 0.000
(3,1) vs. (0,1) 30.74467 2.998826 10.25 0.000
(1,1) vs. (1,0) -0.741 2.998826 -0.25 1.000
(2,0) vs. (1,0) 8.147334 2.998826 2.72 0.000
(2,1) vs. (1,0) 5.555 2.998826 1.85 0.001
(3,0) vs. (1,0) 6.666 2.998826 2.22 0.000
(3,1) vs. (1,0) 2.962667 2.998826 0.99 0.001
(2,0) vs. (1,1) 8.888334 2.998826 2.96 0.001
(2,1) vs. (1,1) 6.296 2.998826 2.10 0.001
(3,0) vs. (1,1) 7.407 2.998826 2.47 0.000
(3,1) vs. (1,1) 3.703667 2.998826 1.24 0.001
(2,1) vs. (2,0) -2.592334 2.998826 -0.86 1.000
(3,0) vs. (2,0) -1.481333 2.998826 -0.49 1.000
(3,1) vs. (2,0) -5.184667 2.998826 -1.73 1.000
(3,0) vs. (2,1) 1.111 2.998826 0.37 1.000
(3,1) vs. (2,1) -2.592333 2.998826 -0.86 1.000
(3,1) vs. (3,0) -3.703334 2.998826 -1.23 1.000
46
Appendix 5e. Two way ANOVA results for the Memory Retention Phase
N=240 R-squared = 0.3066

Root MSE = 17.579 Adj R-squared = 0.2857
Factor Partial SS df MS F Prob > F

Bacopa monnieri 27098.3333 3 9032.77778 29.23 0.0000
Sleep deprivation 3375 1 3375 10.92 0.0011
Bacopa + sleep deprivation 1231.66667 3 410.555556 1.33 0.2658
Appendix 5f. Pairwise Comparisons of Marginal Linear Predictions between the control and
treatment groups for the Memory Retention Phase
Factor
Bacopa monnieri conc. Contrast Standard Error t P > |t|
low vs. none 9.333333 3.209481 2.91 0.024
low vs. mid 25.66667 3.209481 8.00 0.000
low vs. high 24 3.209481 7.48 0.000
mid vs. none 16.33333 3.209481 5.09 0.000
mid vs. high 14.66667 3.209481 4.57 1.000
high vs. none -1.666667 3.209481 -0.52 0.000
Sleep Deprivation
slpD vs. normal -7.5 2.269446 -3.30 0.001
Appendix 5g. Pairwise Comparisons of Marginal Linear Predictions for the Memory Retention
Phase Using Bonferroni Method
x1=B. monnieri treatment groups (0= none, 1= low, 2= mid, 3= high)
x2=sleep deprivation groups (0=normal, 1=slpD)
Treatment Groups (x1,x2) Contrast Std. Error t P > |t|

(0,1) vs. (0,0) -12.66667 4.538891 -2.79 0.000
(1,0) vs. (0,0) 8.666667 4.538891 1.91 0.000
(1,1) vs. (0,0) -2.666667 4.538891 -0.59 1.000
(2,0) vs. (0,0) 20.66667 4.538891 4.55 0.000
(2,1) vs. (0,0) 18 4.538891 3.97 0.003
(3,0) vs. (0,0) 19.33333 4.538891 4.26 0.001
(3,1) vs. (0,0) 16 4.538891 3.53 0.014
(1,0) vs. (0,1) 21.33333 4.538891 4.70 0.000
(1,1) vs. (0,1) 10 4.538891 2.20 0.000
(2,0) vs. (0,1) 33.33333 4.538891 7.34 0.000
47
(2,1) vs. (0,1) 30.66667 4.538891 6.76 0.000

(3,0) vs. (0,1) 32 4.538891 7.05 0.000
(3,1) vs. (0,1) 28.66667 4.538891 6.32 0.000
(1,1) vs. (1,0) -11.33333 4.538891 -2.50 0.001
(2,0) vs. (1,0) 12 4.538891 2.64 0.000
(2,1) vs. (1,0) 9.333333 4.538891 2.06 0.001
(3,0) vs. (1,0) 10.66667 4.538891 2.35 0.000
(3,1) vs. (1,0) 7.33333 4.538891 1.62 0.001
(2,0) vs. (1,1) 23.33333 4.538891 5.14 0.000
(2,1) vs. (1,1) 20.66667 4.538891 4.55 0.000
(3,0) vs. (1,1) 22 4.538891 4.85 0.000
(3,1) vs. (1,1) 18.66667 4.538891 4.11 0.002
(2,1) vs. (2,0) -2.666667 4.538891 -0.59 1.000
(3,0) vs. (2,0) -1.333333 4.538891 -0.29 1.000
(3,1) vs. (2,0) -4.666667 4.538891 -1.03 1.000
(3,0) vs. (2,1) 1.333333 4.538891 0.29 1.000
(3,1) vs. (2,1) -2 4.538891 -0.44 1.000
(3,1) vs. (3,0) -3.333333 4.538891 -0.73 1.000
Appendix 5h. Analysis of Covariance (ANCOVA) results between Average Pass Rates of
Learning and Memory Retention
MR Learning
MR 1.000
Learning 0.4739 1.000
48

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Effects of Bacopa monnieri Supplementation on the Learning and Short-Term Memory Retention

of Sleep-Deprived Drosophila melanogaster

Bradley Ashley G. Ong

In partial fulfillment of the requirements

BRADLEY ASHLEY GUE ONG

For the degree of

1:00 P.M., 13 April 2016

THESIS ADVISER THESIS CO-ADVISER

THESIS READER THESIS READER

Endorsed by: Authorized by:

Miriam P. de Vera, Ph.D. Elena M. Ragragio, MA Sci. Educ.

The thesis attached hereto, EFFECTS OF Bacopa monnieri SUPPLEMENTATION

MIRIAM P. DE VERA, Ph.D. PAUL MARK B. MEDINA, Ph.D.

MELODY ANNE B. OCAMPO, M.S. MA. CRISTINA A. BORDALLO, M.P.H.

This undergraduate thesis is hereby officially accepted as partial fulfillment of the

ELENA M. RAGRAGIO, MA Sci. Educ. LEONARDO R. ESTACIO Jr., Ph.D.

II. Educational Background

Secondary Jubilee Christian Academy, Quezon City, Philippines

III. Collegiate Organizations

Member (2014-2016), U.P. Manila Biorhythm

Maria Clarice N. Villanueva

II. Educational Background

III. Collegiate Organizations

 the Lord Almighty, for the strength and everything in between.

Bradley Ashley G. Ong Maria Clarice N. Villanueva

Title Page ................................................................................................................................ …..i

Figure 1. Survival rate of Drosophila melanogaster on the difference of 10-fold concentration

Appendix 2. T-Maze Setup for Aversive Phototaxis Suppression Assay…………………….….…..44

Appendix 3. Sleep Nullifying Apparatus………..…………………………………………….....45

Appendix 4. Methodology flowchart…………………………………………………………….46

Appendix 5. ANOVA Results and Post Hoc Results…………………………………………….47

Aversive phototaxic suppression (APS) is a behavioral assay in which photopositive fruit

Background of the Study

leading to decrements in cognition as well as in psychomotor vigilance response speed or the

in impaired subsequent retention (Yoo, 2007).

and operant conditioning (Tully and Quinn, 1985).

Statement of the Problem

term memory (STM) retention of non-sleep-deprived and sleep-deprived Drosophila

on the learning capacities and STM retention in non-sleep-deprived and sleep-deprived D.

among non-sleep-deprived and sleep-deprived D. melanogaster with supplementation.

Significance of the Study

information on the effects of B. monnieri supplementation on non-sleep-deprived and sleep-

deprivation, and night shift-requiring jobs.

Scope and Limitations of the Study

predicted, inferred and related to the collected data through literature.

REVIEW OF RELATED LITERATURE

Behavioral Learning Assays

altered when subjected to experimental behavioral-genetic methods. Such procedures include

quantitative screening, mathematical analyses of heritability and selective breeding performed

the science of neurogenetics followed suit (Vosshall, 2007).

Research attention in phototaxis, however, gradually faded in favor of olfactory-based

olfactory-based memory involved a series of experiments: chemical ablation, induced genetic

mutation and radiofrequency ablation (de Belle and Heisenberg, 1994).

less known than those through olfaction (van Swinderen, 2011).

While olfactory-based behavior of Drosophila is continually expounded through the

knowledge of mushroom bodies, the physiological basis of visual-based behavior is poorly

used in several experiments on neurobiological behaviors of Drosophila. Quinine is used as a

In aversive phototaxic suppression (APS) assay, an individual fly is operantly conditioned

Seugnet et al., 2009).

Effects of Sleep Deprivation on Behavior

in learning and memory.

Resting Drosophila is defined as a long period of immobility and an increased arousal

fruit flies (Nitz et al., 2002).