NEURO
article
Genesis
Learning and non-learning effects of Ginkgo
biloba extract EGb 761 in Aplysia californica
Mary Petrosko1 and Robert Calin-Jageman2
1
Department of Psychology, Dominican University
Correspondance should be addressed to Mary Petrosko (petr0308@umn.edu)
2
Department of Neuroscience, Dominican University
SUMMARY: Existing research about Ginkgo’s ability to improve memory is mixed, and the neural mechanism by which
it may affect behavior has yet to be determined. To explore claims of efficacy, the behavioral and physiological effects of
Ginkgo extract EGb 761 (Schwabe Pharmaceuticals) were observed here in Aplysia californica. The effects of EGb 761 were
measured on spontaneous activity, reflex sensitivity, rates of learning and forgetting in a long-term habituation paradigm
and subsequently in spontaneous and siphon-evoked central nervous system (CNS) activity. Effects were measured after
acute and long-term exposure to EGb 761. No significant effects of EGb 761 were observed on any of the dependent measures, suggesting that the EBg 761 compound is not bioactive in the Aplysia CNS. These findings suggest the following: 1)
EGb 761’s mechanism of action is outside of the CNS (e.g. cerebral blood flow); 2) its mechanism of action is not conserved
across the animal kingdom; or 3) that EGb 761 is not bioactive.
Supplements to improve learning and memory abound
in supermarkets and health food stores. These supplements are not regulated by the Food and Drug Administration and do not undergo testing to verify their efficacy.
Rigorous and controlled testing must to be conducted to
determine the efficacy and safety of the supplements.
A popular learning and memory supplement is Ginkgo biloba, which has been used historically for medicinal purposes. The patented extract EGb 761, marketed
by Schwabe Pharmaceuticals (Karlsruhe, Germany),
contains a formula of 24% flavones and 6% terpenoides derived from the ginkgo plant (DeFeudis & Drieu,
2000). This formula has been marketed as the most ‘effective’ extract of ginkgo (Muller & Chatterjee, 2003). The
purported memory effects of ginkgo have been supported
by the argument that it increases circulation in the brain
and has antioxidant properties (DeFeudis & Drieu, 2000;
Gold, Cahill & Wenk, 2003; Petkov, Belcheva & Petkov,
2003). The formula of flavones and terpenoides in EGb
761 are thought to enhance the antioxidant and circulatory properties of the extract. For the purposes of this paper, “EGb 761” will identify the ginkgo extract patented
by Schwabe Pharma. Any other extract of ginkgo will be
referred to as “ginkgo extract.”
Evidence of efficacy
The learning and memory effects of ginkgo have
been investigated in humans and other mammals, with
most studies focusing on the effect of EGb 761. Despite
several studies investigating the drug, the literature presents mixed conclusions regarding the efficacy of gingko
supplements. Evidence does exist for EGb 761 effectiveness in alleviating learning and memory deficits in human and animal subjects (Kanowshi, Hermann, Stephan,
Wierich & Horr, 1996; Lin, Cheng, Hsu & Chang, 2003;
14 | neurogenesisjournal.com | Fall 2012 | Vol 2 Issue 1
Tchantchau, Xu, Wu, Christen & Luo, 2007; Wang,
Wang, Wu & Cai, 2006); on the other hand, some studies
report opposing results (Birks & Grimley, 2009; Dekosky
et al., 2008). However, there is consensus regarding EGb
761 efficacy in subjects with normally functioning brains;
in these populations, EGb 761 provides no improvement
in memory (Burns, Bryan, & Nettelbeck, 2006; DeKosky
et al., 2008; Solomon et al., 2002).
The learning and memory effects of ginkgo have been
investigated in humans and other mammals, with most
studies focusing on the effect of EGb 761. Despite several studies investigating the drug, the literature is mixed
regarding its efficacy. Studies in human and animal subjects with brain abnormalities has supported EGb 761’s
effectiveness in alleviating deficits in learning and memory (Kanowshi, Hermann, Stephan, Wierich & Horr,
1996; Lin, Cheng, Hsu & Chang, 2003; Tchantchau, Xu,
Wu, Christen & Luo, 2007; Wang, Wang, Wu & Cai,
2006); however, other studies have failed to support this
effect (Birks & Grimley, 2009; Dekosky et al., 2008).
In normally functioning brains, however, most research
indicates that EGb 761 provides patients with no clear
improvement in memory (Burns, Bryan, & Nettelbeck,
2006; DeKosky et al., 2008; Solomon et al., 2002).
In rodent animal models, ginkgo has demonstrated
selective influence on learning and memory. In studies
with older or impaired rats, EGb 761 has been shown
to be relatively effective in alleviating learning deficits
(Kanowshi et al., 1996; Lin et al., 2003; Tchantchau et
al., 2007; Wang et al., 2006). Lin et al. (2003) examined the effect of ginkgo in rats with bilateral common
carotid artery ligation, which produced chronic cerebral
insufficiency. EGb 761 improved impairments in spatial
learning and motor function more effectively than an
extract of local ginkgo. However, it is important to note
article
that performance did not return to normal function, and
impairments in spatial learning persisted beyond EGb
761 treatment . Wang et al. (2006) found that EGb 761
administered to older rats improved their spatial learning
for a maze task. Furthermore, they found that hippocampal long-term potentiation (LTP) was increased in
subjects treated with EGb 761. This increase in LTP may
account for memory improvements in treated subjects,
as the hippocampus is critical in forming new memories
and spatial learning.
Research on gingko efficacy in humans with cognitive
deficits is less conclusive. In a large, double-blind, placebo-controlled study conducted by Le Bars et al. (1997), a
slight but consistent memory improvement was observed
in participants with dementia on gingko supplementation. Participants treated with EGb 761 performed better
on the Alzheimer’s Disease Assessment Scale-Cognitive
subscale and on the Geriatric Evaluation by Relative’s
Rating Instrument. However, the Ginkgo Evaluation of
Memory (GEM) by DeKosky et al. (2008) found that
EGb 761 failed to reduce the incidence of dementia in
older adults (≥75 years old). GEM is a large and wellcontrolled study supported by the National Institutes of
Health to test the efficacy of EGb 761 as a preventative
treatment for Alzheimer’s disease. The study tracked participants for an average of five years and found that EGb
761 failed to prevent or delay the incident of dementia
in older adults. Based on these conflicting results, EGb
761 may work through a mechanism which improves
memory in those who have developed dementia but is
not involved with pre-dementia memory mechanisms.
While results are contradictory in impaired populations, similar studies with normal populations have
shown little overall effectiveness of the ginkgo extract.
Solomon, Adams, Silver, Zimmer, and DeVeaux (2002)
examined the effect of ginkgo extract in healthy individuals aged 60 years or older during six weeks of supplementation. No measurable learning, memory, or attentional
improvements were found for treated individuals. Similarly, no significant learning or memory improvement
occurred for a younger population of participants--aged
18 to 43 years--observed across twelve weeks of gingko
extract supplementation (Burns, Bryan, & Nettelbeck,
2006). In addition, the studies that do report disparate
positive effects of EGb 761 in healthy adults, their conclusions may be statistically unreliable because of small
sample sizes and unclear outcome measures (Birks &
Grimley, 2009; Gertz & Kiefer, 2004).
Outstanding questions
Overall, the existing evidence suggests that EGb 761
may have neuroprotective qualities but that it does not
actively improve learning or memory in normally functioning brains. One limitation with existing research,
NEURO
Genesis
however, is the focus on a single indicator of efficacy:
long-term recall measured at a single time point. This
measure of memory and learning function may not be
sensitive to changes in a normal population. To more
fully evaluate the effects of EGb 761 in normal populations, it may be useful to collect a broad range of learning
indicators, including rate and depth of learning, shortterm memory performance, and the duration of longterm memory.
Another limitation in the study of gingko extract is
our inability to identify biological mechanisms, which
may be linked to the behavioral effects of EGb 761. One
suggested mechanism by which EGb 761 may operate
lies outside of the CNS; EBg 761 may improve blood
circulation and increase the flow of oxygen to the brain
(DeFeudis & Drieu, 2000). Other evidence points to the
direct effects of EGb 761 in the CNS; older rats treated
with EGb 761 exhibited increased LTP in vivo (Wang et
al., 2006). Similarly, mice brains with applied EGb 761
exhibited increased LTP and neuronal excitatibility in vitro (Williams, Watanabe, Schultz, Rimbach, and Krucker,
2004). Additional studies have also found that EGb 761
increases neuronal plasticity, and protects against neuronal apoptosis in cell cultures (Defeudis & Drieu, 2000;
Luo et al., 2002; Smith et al., 2002). Despite these findings, researchers have not yet explored the neural mechanisms by which EGb 761 influences behavior.
Aplysia as a model system
The marine gastropod, Aplysia californica (Figure
1), is a useful model organism in which to evaluate the
effects of EGb 761. Aplysia provide sensitive measurements of the effects of EGb 761 effects in a biological
system that can more easily be connected to behavioral
measures. Aplysia have long proven useful for identifying
basic mechanisms of learning and memory. One attractive feature is that Aplysia have a relatively small number
of nerve cells, numbering less than 10,000 in its CNS
(Kandel, 2001). Moreover, Aplysia have large nerve cells
(up to 1 mm in diameter), which are both easy to monitor and manipulate (Moroz et al., 2006). These features
make Aplysia uniquely useful for relating behavioral
and neural function. As a result of their relatively simple
neural circuitry, the sensory and motor neural circuit has
been identified and studied extensively, relating its activity to the behavior it elicits (Kandel, 2001). In addition,
the simple neural circuitry allows for extracellular and
intracellular recording, with which neural activity can
be analyzed and related to behavioral responses. Aplysia
have been used to understand basic physiological mechanisms of habituation, sensitization, operant conditioning, place conditioning, and other forms of learning and
memory (Brembs, 2003; Glanzman, 2006; Rankin, 2002;
Stopfer & Carew, 1996).
Vol 2 Issue 1 | Fall 2012 | neurogenesisjournal.com | 15
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Genesis
Stimulus
30 stimuli, 1 min ISI
1 day
Pre-test
Training
Post-test
Figure 1: Procedure for Experiment 1, to elicit long-term habituation of S-SWR.
Animals were assessed for baseline responding with the pretest, 3 stimuli administered at 10 minute ISI. Animals were then trained in 5 sessions, each session
consisting of 30 stimuli at 1 minute ISI, with 24 hours between each session. After training, short- and long-term retention post-testing were administered in
the same way as the pretest. Post-testing was administered 1 hour after the last training session, then once a day for the subsequent 6 days.
Current study
To address the diverse and discordant literature on
EGb 761, this study used a three-part approach, which
explored possible learning, arousal, and physiological
effects of EGb 761 on acute and long-term exposure
in Aplysia. The three experiments included measures
on multiple time scales and explored possible effects on
different behavioral and physiological outcome measures. The combined results offer a sensitive and inclusive
analysis of possible behavioral and physiological effects of
EGb 761.
Effect of EGb 761 on long-term learning
The first experiment examined the effect of EGb 761
on long-term habituation after long-term exposure. Habituation is a commonly used model of learning in Aplysia; it is a type of learning in which a subject’s response to
a stimulus decreases as a result of repeated presentation
of the stimulus. Although simple, habituation shares
many attributes with more complex learning mechanisms (Carew, Pinsker, & Kandel, 1972; Stopfer, Chen,
Tai, Huang, & Carew, 1996; Zolman & Peretz, 1987).
Like more advanced learning mechanisms, habituation
response rates can improve with training, are sensitive to
the pattern and frequency of training, and can produce
both short- and long-term memories (Abramson, 1994).
A benefit of studying habituation is that it provides
multiple indicators of learning and memory. This first
experiment examined four separate indicators: depth of
learning, rate of learning, short-term memory retention,
and long-term memory retention.
The behavior used for habituation training in this
experiment was the siphon-elicited siphon withdrawal
reflex (S-SWR). The S-SWR is a simple reflex in which
the siphon is withdrawn into the mantle after mild stimulation to the siphon (Carew et al., 1972). The S-SWR
16 | neurogenesisjournal.com | Fall 2012 | Vol 2 Issue 1
is easily observable and is easily quantified as the time
from when the animal withdraws its siphon to the time
it begins to relax the siphon back to the resting position.
In the S-SWR, habituation can be elicited by repeatedly
stimulating the siphon, producing a gradual decrease in
the duration of the SWR. Depending on the pattern
and duration of stimulation, it is possible to produce
both short- and long-term habituation (Carew et al.,
1972; Stopfer et al., 1996; Zolman & Peretz, 1987). This
experiment explored the possible effects of long-term
EGb 761 exposure on four separate learning and memory
indicators of long-term habituation of the S-SWR.
Effect of EGb 761 on arousal after acute exposure
The second experiment explored the effect of EGb
761 on arousal and gross motor movement after acute
exposure to EGb 761. Past research suggests that significant effects can be elicited from manipulations other
than long-term gingko exposure; a single dose of EGb
761 before the administration of a narcotic resulted in
increased arousal in treated animals (Brochet, Chermat,
DeFeudis, & Drieu, 1999). Additional studies in humans
have shown a slight improvement in reaction times on
short-term memory tasks and sustained attention following acute exposure to EGb 761 (Elsabagh, Hartley, Ali,
Williamson, & File, 1995; Kennedy, Scholey, & Wesnes,
2000; Rigney, Kimber, & Hindmarch, 1999).
To test possible arousal effects of EGb 761, the second
experiment examined behavioral measures of locomotion
and S-SWR after acute exposure to EGb 761. Previous
research in Aplysia has used both locomotion and SSWR as indicators of arousal after exposure to a treatment (Marinesco, Wichremasinghe, Kolkman, & Carew,
2004). Together, these measures serve as sensitive indicators of acute animal arousal.
NEURO
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Genesis
Effect of EGb 761 on CNS activity
The third experiment examined the physiological affect of EGb 761 on background and nerve-evoked neural
activity in the CNS after acute exposure. An investigation of the possible biological mechanism by which EGb
761 affects the organism followed. Research with rats
and mice has shown that EGb 761 can increase hippocampal activity and LTP when administered in vivo and
in vitro (Lanahan, Lyford, Stevenson, Worley, & Barnes,
1997; Wang et al., 2006; Williams et al., 2004). In vitro
studies have shown an increase in neuronal excitability
and synaptic transmission when EGb 761 was applied to
mice brain slices (Williams et al., 2004). This evidence
indicates a correlation between EGb 761 and the neural
mechanisms of memory. As Aplysia offer a simple neural
circuit from which the activity of the CNS can be easily
monitored and quantified, this study directly tested the
claim that EGb 761 affects the CNS.
The three experiments performed here allowed for a
multi-dimensional analysis of the effects of EGb 761 on
multiple behavioral and physiological outcome measures;
such a comprehensive evaluation of the possible effects of
EGb 761 in a model system may provide a link between
physiology and behavior.
Experiment 1
A number of Aplysia behaviors have been analyzed at
the neural level. This particular study explored the effect
of EGb 761 on long-term habituation of the S-SWR. To
produce long-term decreases in S-SWR behavior, Aplysia received five separate training sessions consisting of
30 stimuli applied to the siphon, each eliciting a S-SWR.
Training sessions were administered daily for five days
(Figure 2). This protocol has shown significant long-term
habituation learning lasting up to 14 days after training
(Stopfer et al., 1996).
Multiple indicators of learning and memory were
assessed: depth of learning, rate of learning, short-term
retention, and long-term retention. Depth of learning
was measured as the change from baseline to minimum
responding during training. Rate of learning indicated
how fast the animal habituated and was measured as
the number of trials during training to reach minimum
responding. Short- and long-term retention was assessed by comparing each animal’s baseline responding
to posttest responding, indicating the amount of learning
retained after training. Together, these measures provide a
sensitive composite of learning and memory outcomes.
By using four separate learning measures, this study
improves upon the standard reliance on one measure of
memory at a single point in time and may reliably detect
subtle changes in learning performance.
Method
Subjects
We used 24 Aplysia californica. Animals (late juveniles, 75-80g) were purchased from the NIH Aplysia resource facility (Miami, FL) and housed in separate plastic
containers in a 15° C tank of aerated artificial seawater
(ASW). All animals were fed a diet of dried seaweed,
delivered three times a week. Aplysia were maintained at
a constant 12:12 light/dark schedule (light 6 am–6 pm,
dark 6 pm – 6 am), and underwent treatment and training during light cycles.
Drug administration
Animals were randomly assigned to one of the three
experimental groups (n=8): control, low dose and high
dose. Animals were pre-treated with EGb 761 for ten
days prior to training. The Gingko biloba supplement
used was powdered EGb 761 containing 24% ginkgo flavone glycosides and 6% terpene lactones (Schwabe Pharmaceuticals, Germany). The supplement was dissolved
8
High
7
Low
Control
Seconds
6
5
4
3
2
1
0
BL TR 1
TR 2
TR 3
TR 4
TR 5
Figure 2: Long-term habituation learning curve data between experimental groups.
S-SWR (s) throughout habituation training. Significant effect of habituation training, F(4, 84) = 8.02, p < .01. No significant effect of
ginkgo on the rate or magnitude of learning (F < 1).
Vol 2 Issue 1 | Fall 2012 | neurogenesisjournal.com | 17
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Genesis
in an incubation chamber containing 2 liters of ASW.
Treated animals were soaked in the incubation chamber for 1 hour each day; control animals were soaked in
untreated ASW. Animals were housed and trained in
their home tank throughout the experiment and were not
handled at any time.
For the low-dose group, EGb 761 was dissolved in the
incubation tank in quantities that achieve a 0.17% solution. When equilibriated, each tank provided a peak dose
of 0.133 mg for each 75g animal, or 1.77 mg/kg. The
high-dose group received twice the low-dose exposure:
incubation in a 0.35% solution providing a peak dose
of 3.54 mg/kg. The solution was mixed by an outside
researcher, and the researcher responsible for habituating and timing responses was blind to the experimental
groups.
S-SWR response
The S-SWR behavior was quantified as the duration
of the withdrawal reflex. A researcher manually stimulated the Aplysia by placing a thin, plastic stimulator against
the inner wall of the siphon. The stimulator is then drawn
up along the siphon rapidly to elicit the SWR in the
mantle shelf. S-SWR behavior was measured in seconds
by the researcher after administering the stimulation.
Timing began once the siphon was contacted and ended
once the siphon began to relax to its normal position.
Habituation training
Baseline responsiveness was determined as the mean
response to three stimuli administered at a ten minute
interstimulus interval (ISI). A ten minute ISI does not
produce habituation. Habituation training consisted of
five sessions of 30 stimuli administered at a 1 minute ISI
with a 24-hour interval between training sessions (Figure
1).
Memory tests
After habituation training, animals were tested for
both short- and long-term habituation. Short-term
testing occurred one hour after the final training session
7
6
Seconds ± SEM
5
4
3
2
1
0
1 hr
1 Day
2 Day
3 Day
4 Day
5 Day
6 Day
Figure 3: Forgetting curve for 6 days post habituation training between
experimental groups.
Average S-SWR response between groups. Significant effect of forgetting,
F(5, 105) = 4.38, p < .01. No significant effect of ginkgo on the rate or
magnitude of forgetting (F < 1).
18 | neurogenesisjournal.com | Fall 2012 | Vol 2 Issue 1
on the fifth day of training. Long-term testing occurred
once a day for six days following the final training day.
Each memory test consisted of three siphon stimulations
with a 10 minute ISI.
Statistical approach
Data were analyzed using the Statistical Package for
the Social Sciences (SPSS Inc., Chicago, IL, USA). The
basic strategy was to compare task performance between
treatment groups (control, low, and high). Univariate
analyses of variance (ANOVA) assessed for group differences. Alpha levels below 0.05 were considered statistically significant.
Results
Baseline responding
No significant effect of EGb 761 on baseline responding was found. After ten days of pre-treatment, baseline
responding was characterized by timing three elicited
S-SWRs at ten minute intervals in all three treatment
groups (control, low, and high dose of EGb 761). S-SWR
responsiveness was similar across treatment groups: control (M = 6.41, SD = 3.72), low (M = 7.03, SD = 2.72),
and high (M = 7.79, SD = 4.60) treatment level (Figure
3). This result was confirmed with a one-way betweengroups analysis of variance (ANOVA); no significant
main effect of treatment condition (F(2, 21) = .27, p >
.05) was observed. Repeated exposure to EGb 761 did
not affect baseline S-SWR. Any difference in learning or
memory measures between experimental groups would
not be due to unequal initial conditions between experimental groups, but instead due to experimental condition.
Learning effects
A mixed-factor ANOVA demonstrated that the
means between the drug conditions were not significantly
different, F(2, 21) = .97, p > .05, during any of the five
training sessions (Figure 3). Therefore, animals in each of
the three drug conditions acquired habituation at a similar rate, and EGb 761 did not affect rate of learning.
A one-way between-groups ANOVA was conducted to
evaluate the main effect of EGb 761 on average S-SWR
at the end of last day of training. No significant differences were found between the conditions, F(2,21) =
.97, p > .05 (Figure 2). This result is expected because
no significant differences in rate of learning were found
between the three conditions.
All groups were given habituation training of 1 training session per day for 5 days (20 stimuli per session, 1
minute ISI). By the end of the training, S-SWR responsiveness had decreased substantially across all groups:
control (M = 2.85, SD = .79), low (M = 2.92, SD = 1.55),
and high (M = 2.92, SD = .51) treatment level (Figure
3). To evaluate the main effect of habituation training,
average S-SWR across the last 5 trials in each training
session were analyzed with a repeated measures ANOVA.
NEURO
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Genesis
Stimulus
30 min
ASW
1 day
Gingko
Figure 4: Procedure for experiment 2 to determine acute arousal effects
of EGb 761. S-SWR measures were taken at the beginning and end of
each session, 30 min ISI. Animals were exposed to EGb 761 during Days
3-4 of the experiment.
The ANOVA indicated a significant training effect,
F(4, 84) = 8.02, p < .01. Follow-up polynomial contrasts
indicated a significant linear effect, with response means
decreasing over time, F(1, 21) = 14.64, p < .01.
To more fully understand the shape of the learning
curve, a variety of simple equations were fit to the training data. For this analysis, all trials were included and
data for each drug condition was collapsed because there
was no main effect of drug condition and no interaction between drug condition and training session (F(4,
84) = .77, p > .05). The data best fit to an exponential
function, which accounted for 75% of the variance in
the training data. The parameters for this function were
well constrained by the data: at the end of training, the
animals’ S-SWR was 51.81% of their baseline responding, 95% confidence between 49.74% - 53.88%. After
the first 19.91 stimulations, animals’ response decreased
40.68% from baseline responding. This means the greatest amount of learning typically occurred during the first
training session (which consisted of 20 stimulations).
Memory Effects
No significant group difference was found on S-SWR
one hour after the last training session, F(2, 21) = .96,
p > .05 (Figure 3). This result indicates that the drug
condition did not affect short-term memory retention:
control (M = 3.61, SD = 1.60), low (M = 3.23, SD = .27),
and high (M = 3.77, SD = 1.31). Similarly, no significant
group differences were found on S-SWR for the six days
following the end of training, F(2, 21) = .88, p > .05 (Figure 3). These results failed to support an effect of EGb
761 on short and long-term memory.
To evaluate habituation training forgetting, a one-way
repeated measures ANOVA was conducted to evaluate
all animals’ S-SWR for the six days after training ended.
The ANOVA indicated a significant forgetting effect,
F(5, 105) = 4.38, p < .01 (Figure 3). Therefore, animals
displayed significant forgetting during post-testing.
A two-way mixed factor ANOVA was conducted to
evaluate a possible interaction between EGb 761 condition and the forgetting curve over the six days following
training. The ANOVA failed to support a significant
ASW
interaction between EGb 761 and forgetting, F(5, 105)
= .84, p > .05. Therefore, the results indicate that animals
forgot training at a similar rate regardless of EGb 761
condition.
Experiment 2
In addition to possible learning benefits, EGb 761 has
been purported to have acute arousal effects. Research
indicates that EGb 761 can have an arousal effect in
animals and humans after acute exposure (Brochet et
al., 1999; Elsabagh et at., 1995; Rigney et al., 1999). To
better document this effect in a physiologically tractable
system, this experiment examined the acute arousal effects of EGb 761 in Aplysia. Two measures of arousal
were used: S-SWR sensitivity and locomotion.
Method
Experimental design
Experiment 2 used a within subjects design with all
animals (n = 8) undergoing the same experimental sequence (Figure 4). Animals were transferred to an experimental tank containing 2 liters ASW while remaining in
their home containers and were not handled at any time.
While in the experimental tank, animals were videotaped
to analyze locomotion. Animals were taped once a day
for 30 minutes on 6 consecutive days.
Drug administration
Days 1-2, animals were only exposed to ASW and
were not exposed to EGb 761. Days 3-4, animals were
exposed to twice the recommended dose of Ginkgo. EGb
761 was dissolved in the 2 liters of ASW in the experimental tanks to achieve a 0.35% solution providing a
peak dose of 3.54 mg/kg, double the recommended daily
dose (120 mg recommended daily dose, average adult
weights 67.5 kg, providing a dose of 1.77 mg/kg). Days
5-6, animals were only exposed to ASW and were not
exposed to EGb 761.
S-SWR response
S-SWR response was measured in seconds by the
researcher after administering the stimulation manually,
as in Experiment 1. Each animal’s S-SWR was recorded
once at the beginning of videotaping and a second time
at the end of taping, 30 minute inter-stimulus interval
(ISI).
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Crawling
The animals’ amount of crawling was analyzed using
SwisTrack software (Lochmatter et al., 2008). SwisTrack provided the amount of pixels each animal moved
during the 30 minute recording period by tracking their
positions on an XY coordinate. Animal positions were
calculated at an interval of 5 times per minute. The total
number of pixels moved during recording was calculated
using the Pythagorean Theorem. The distance moved
in pixels was added together to generate a total crawl
amount. The total pixels each animal moved was converted to the equivalent cm moved (Figure 5). The pixel to
cm conversion was calculated using a calibration frame of
the video; the amount of pixels generated was compared
to the specified distance moved by a sample object.
Results
S-SWR response
S-SWR was compared between EGb 761 exposed
and non-exposed conditions. The within-subject control
was highly reliable. S-SWR responses in each animal
were very stable across days, alpha = 0.86 (Figure 6). A
within-subjects ANOVA comparing the animals’ S-SWR
behavior during pre-exposure, exposure, and postexposure days showed no significant effect of EGb 761:
pre-exposure (M = 7.25, SD = 2.17), exposure (M = 6.44,
SD = 2.39), and post-exposure (M = 7.01, SD = 2.16),
F(2, 14) = .98, p > .05.
Crawling
The amount of crawling between the two conditions
(non-exposed, EGb 761 exposed) was compared to detect
a significant effect of ginkgo. A within subjects ANOVA
comparing the animals’ total movement during treatment
condition showed no significant effect of EGb 761on
cm crawled: pre-exposure (M = 1441.28, SD = 1352.19),
exposure (M = 2027.82, SD =1068), and post-exposure
(M = 1632.22, SD = 1172.38), F(2, 14) = .57, p > .05
(Figure 7). However, the amount each animal crawled
varied substantially between days, and the within-subject
control was not reliable, alpha = 0.20 (Figure 7 and 8).
Experiment 3
While EGb 761 failed to produce an effect in the
Video Recording
1
3
Movement Tracking
2
1
2
3
4
4
Figure 5: Example SwisTrack locomotion tracking of 4 Aplysia during
30 min video recording session.
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behavioral measures tested in Experiments 1 and 2, there
is the possibility that it could have a direct effect on the
CNS which is not manifested behaviorally. The biological
mechanism by which EGb 761 is purported to operate is
unclear. Some research indicates that it operates outside
of the CNS, working through blood flow, etc. (DeFeudis, 2000). Other research, however, with other animal
models and humans indicates that EGb 761 can increase
neuronal activity and hippocampal function in subjects
both in vivo and in vitro (Lanahan et al., 1997; Wang et
al., 2006; Williams et al., 2004). A possible mechanism
by which EGb 761 can affect the organism is through direct influence in the CNS. Neuronal activity and synaptic
transmission has increased in some studies which look at
the effect of EGb 761 when directly applied to the brain
(Lanahan et al., 1997; Williams et. al., 2004).
Experiment 3 tested the direct effects of EGb 761 on
the spontaneous and nerve-evoked activity in the CNS.
Nerve activity during three conditions (pre-exposure to
EGb 761, exposure, and post-exposure) and was assessed
by two measures. First, background nerve activity was
measured as the normal spontaneous activity occurring in
the CNS during the experiment. Second, nerve-evoked
evoked activity was measured after an electrical shock
was administered to a sensory nerve.
The 1 minute preceding and following each nerve
shock were excluded from background nerve analysis.
Each minute segment was compared as a percentage of
baseline responding (nerve activity during first 5 minutes
of recording).
Method
Animal preparation
Aplysia (n = 5) were anesthetized (50 mL MgCl) and
dissected according to established protocol (Stopfer et al.,
1996). The CNS (right and left P9 sensory nerves, ring
ganglion, right and left connective nerves, abdominal
ganglion, and siphon motor nerve) was removed from the
animal and maintained in a 5 mL recording dish with
profused ASW. The CNS was revived with ASW and
rested 1 hour before recording.
Electrophysiology
Nerve activity was recorded extracellularly using suction electrodes with flexible Tygon tips. Nerve recordings were taken for the right P9, right connective, and
siphon nerve in each animal (10hz high-pass filter, 3khz
low-pass, 10k gain, A-M Systems AC/DC Differential
Amplifier model 3000). Nerve recordings were quantified
as the integral of the absolute value of the nerve trace (20
K/s sampling rate).
Spontaneous background nerve activity
Background nerve activity was calculated in one minute segments as the average of the nerve trace integral.
The 1 minute proceeding and following each nerve shock
were excluded from background nerve analysis.
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Genesis
10
Seconds SEM
8
6
4
2
0
1
2
3
Day
4
5
6
Figure 6: S-SWR in experiment 2.
Responses were stable (alpha = .86). No significant effect of EGb 761 on S-SWR (F < 1).
400
CM SEM
300
200
100
Figure 8: Example of SwisTrack locomotion tracking for one
Aplysia across the six experimental days.
0
1
2
3
4
5
6
Day
Figure 7: Average locomotion (cm) by day for experiment 2.
Responses were not stable across days (alpha = .20). No significant effect of EGb 761 on locomotion (F < 1).
10 min
ASW
Gingko
ASW
Figure 9: Procedure for Experiment 3.
This is used to determine background and nerve-elicited nerve activity in the Aplysia central nervous
system (right P9 sensory nerve, right connective nerve, siphon motor nerve).
200µV
Figure 10: Sample of spontaneous nerve activity in and out of EGb 176 bath application.
Nerve-elicited nerve activity
During nerve-elicited nerve activity trials, an
80-90 µA pulse (0.25 s duration, biphasic) was
administered to the left P9 sensory nerve (A-M
Systems Isolated Pulse Stimulator model 2100).
The stimulus artifact (0.5 s after administration
of pulse) was subtracted out of nerve-elicited
nerve activity analysis. Nerve-elicited nerve
activity was calculated as the average of the nerve
trace integral for the 0.3 s following the stimulus
artifact (0.5 s – 0.8 s after pulse administration).
This protocol was validated by comparing SWR
with nerve activity in a set of reduced siphon+tail
preperations; the average of the nerve trace integral for the 0.3 s following the stimulus artifact
(0.5 s – 0.8 s after pulse administration) was
highly correlated with behavioral SWR response.
Average nerve response after each pulse was
compared as a percentage of baseline responding
(nerve activity during first 2 pulses).
Drug administration
During the drug exposure condition, EGb
761 was added to the ASW in the recording dish
(5µL EGb 761/5 mL recording dish capacity).
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Genesis
Experimental design
Recordings were taken for the right P9 sensory nerve,
right connective nerve, and siphon motor nerve continuously for 90 minutes. Ten pulses (80-90 µA) were administered to the left P9 sensory nerve throughout recording
10 minute ISI (Figure 9). For minutes 0-19 the CNS
was exposed to ASW, establishing baseline background
neural activity and nerve-elicited activity. During minutes 19-51 EGb 761 was directly applied to the CNS.
After the 30 minutes of exposure, the profusion tube was
reopened, washing out the EGb 761. For minutes 51-90
post-exposure, the CNS was exposed to ASW during
post-test background activity and nerve-elicited activity
recording.
Results
Background nerve activity
A one-way within-subjects ANOVA was conducted
for each nerve (right P9, right connective, siphon), comparing each nerve’s background activity across treatment
conditions (pre-exposure to EGb 761, exposure, postexposure). EGb 761 demonstrated no significant effect
on total background nerve activity (Figure 10): right P9:
F(2, 8) = .50, p > .05; right connective: F(2, 8) = 1.06, p >
.05; siphon: F(2, 8) = 2.22, p > .05 (Figure 11). Therefore,
background nerve activity was not significantly different
on any of the three nerves between the ASW and EGb
761 conditions.
A one-way within-subjects ANOVA comparing each
nerve’s nerve-elicited activity (right P9, right connective,
siphon) across treatment conditions (pre-exposure to
EGb 761, exposure, post-exposure) showed no significant
effect of EGb 761 on nerve-elicited activity (Figure 12):
right P9: F(2, 8) = .88, p > .05; right connective: F(2, 8)
= .83, p > .05; siphon: F(2, 8) = .44, p > .05 (Figure 13).
Thus, EGb 761 did not significantly change the nerveelicited activity in any of the three monitored nerves.
1.5
Percent of Baseline
SEM
R Connective
Siphon
1
R P9
General discussion
This study applied rigorous and controlled testing to
determine the efficacy of the popular learning and memory supplement, Ginkgo biloba extract EGb 761. Previous research is mixed as to EGb 761’s ability to improve
learning and memory (Birks & Grimley, 2009; DeKosky,
et al., 2008; Gertz & Kiefer, 2004; Le Bars et al., 1997;
Tchantchau et al., 2007) in human and animal models.
The first experiment explored the possible learning and
memory effects of long-term exposure to EGb 761 on a
long-term habituation protocol in the Aplysia. Despite
sensitive learning measures which accounted for several
factors of learning and memory (rate, depth, short-term
and long-term retention), EGb 761 failed to produce an
effect in any dependent measure. EGb 761’s purported
effect of arousal after acute exposure was explored in
Experiment 2 (Brochet, 1999; Rigney, 1999). Two arousal
measures, locomotion and S-SWR sensitivity, were tested
after acute exposure to the drug. Again, EGb 761 failed
to produce an effect on any of the dependent measures.
To further explore the suggested mechanisms by
which EGb 761 is purported to work, Experiment 3 tested its effects in Aplysia CNS. Lanahan (1997) and Williams et. al. (2004) have suggested that EGb 761 operates
on the CNS by increasing neuronal activity and synaptic
transmission; however, no main effect of EGb 761 was
found on the spontaneous background and nerve-elicited
nerve activity in the present study. The general conclusion
from these experiments is that EGb 761 is not bioactive
in the Aplysia nervous system.
Possible interpretations
One possible explanation for the lack of positive
results in these experiments is a lack of experimental
power to detect the effects of EGb 761. The measure of
locomotion after acute exposure to EGb 761 showed
high within-subject variability and low power. However,
this interpretations appears unlikely. Numerous other
tested measures of the efficacy of EGb 761 demonstrated
low within-group variability and high levels of internal
reliability. In addition, the three experiments provide
converging and consistent results. The preponderance of
evidence provided by these experiments thus indicates no
0.5
0
10
20
30
40
50
60
70
80
Time
Figure 11: Background resting neural activity by nerve (P9, right connective, and siphon). EGb 176 does not affect resting neural activity in Aplysia (F<1 for each nerve).
22 | neurogenesisjournal.com | Fall 2012 | Vol 2 Issue 1
200µV
90
1s
Figure 12: Representative siphon nerve recording of average nerveevoked activity as a percent of baseline (first 2 stimulations).
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Genesis
Percent of Baseline
SEM
2
R Connective
Siphon
1.5
1
R P9
0.5
0
0
10
20
30
40
50
60
70
80
90
Time
Figure 13: Nerve-elicited nerve activity as a percent of baseline (activity
after first two pulses) for each nerve (P9, right connective, and siphon).
No significant difference during EGb 761 exposure (F < 1 for each nerve).
effect of EGb 761.
A second possible explanation for the null findings
is that EGb 761 operates by a mechanism in humans
which is not conserved in Aplysia. Aplysia are a useful
animal model to study learning and memory, as their
simple nervous system allows for identification of neural
correlates of behavior. While the simplicity of Aplysia is
an advantage in some respects, this simplicity also limits
the applicability of invertebrate research to mammals.
However, research with Caenorhabditis elegans, another
invertebrate animal model, has indicated beneficial effects of EGb 761 (Luo, 2006; Smith & Luo, 2004; Wu
et al., 2006). Moreover, recent research by Moroz et al.
(2006) to sequence the Aplysia genome indicates that
Aplysia may be more similar to humans than C. elegans
genomically. Because Aplysia have NMDA receptors and
are capable of synaptic plasticity, the animal model has
proven useful for understanding mammalian learning and
memory. Given the strong evolutionary conservation of
the biochemistry of learning, it seems somewhat unlikely
that Aplysia and mammals diverge in their sensitivity to
EGb 761.
Another possible explanation is that EGb 761 works
outside of the CNS and its effects cannot be detected
in Aplysia. Some research suggests that EGb 761 works
through improving blood circulation and increasing the
flow of oxygen to the brain (DeFeudis & Drieu, 2000).
However, there is a lack of evidence to support EGb
761’s effects on blood flow through direct monitoring of
these biological effects. Possible future research can explore this mechanism through direct monitoring of blood
flow and oxygenation as a result of exposure to EGb 761.
A fourth possible conclusion is that EGb 761 is not
bioactive. The study by Solomon et al. (2006) found
that EGb 761 was not active in normal healthy adults.
There have been mixed results as to its effectiveness in
impaired populations; however, the recent GEM study
by DeKosky et al. (2008) concluded that EGb 761
failed to improve memory or prevent dementia in older
adults. A recent meta-analysis also found that EGb 761
was not effective in improving memory in older adults
overall (Birks & Grimley, 2009). Despite the majority of null results in large and well-controlled studies,
mixed evidence to support EGb 761’s beneficial effects
persists. This could possibly be due to publication bias,
in which positive results are favored over negative results
in publication. The proposed benefits of EGb 761 in
positive studies are not consistently demonstrated, and
the inconsistent purported effects do not appear in large,
well-controlled studies. This study with Aplysia fits into
the recent trend of evidence that EGb 761 does not appear to be bioactive in both Aplysia and humans.
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