The Effects of Alcohol on Short Term Memory in Drosophila
Melanogaster
Virginia Brodie, Paul Mascali, Jean Cheng
Manhasset Science Research
January 31, 2010
Abstract
National Institute on Alcohol Abuse and Alcoholism has estimated that
approximately 14 million people in the United States suffer from alcoholism. Alcohol
consumption has long-term effects, including Wernicke-Korsakoff Syndrome, or Alcohol
Dementia, which consists of impaired short-term memory. Alcohol is an organic
compound that with consumption affects the nerve cell of the brain by preventing the
production and propagation of electric impulses. When alcohol is dissolved in the
membrane of memory cells, it disorganizes the membrane, which leads to changes in the
physical and biochemical characteristics. Moore (98) exposed drosophila to ethanol
using an inebrometer, and Miller, tested human’s short term memory and found that short
term memory was weakened. The purpose of this research will be to show how alcohol
consumption affects short-term memory. This will be tested by exposing drosophila to
ethanol regularly and then using olfactory memory to test the short-term memory, by
shocking flies 4 times for a minute over 40 minutes while simultaneously pumping them
an odor. They will then be placed in a chamber where they have the choice to choose the
familiar scent or a new scent, those choosing the new scent are said to have formed short
term memory.
Interest
Alcohol is the most commonly used and widely abused psychoactive drug in the
world. Alcoholism is a disease, which can some times be fatal. Nearly 14 million
Americans meet diagnostic criteria for alcohol use disorders (Grant, 1994), and more than
100,000 U.S. deaths are caused by excessive alcohol consumption each year (Ericson
,2001).
Knowledge Base
Alcohol is an organic compound; its molecular structure is CH3CH2OH. Ethanol,
one of the most common alcohols, is drinking alcohol its molecular formula is C2H6O.
Alcoholism is the continuous or excessive use of alcohol with associated pathologic
results, and affects nearly 8 million Americans. Alcohol affects the nerve cell by
preventing the production and propagation of electric impulses along a network
consisting of axons and synapses. In the brain, one nerve cell communicates information
to many other cells, which in turn receive impulses from many other areas; alcohol
enhances some of these impulses and blunts others. When alcohol is dissolved in the
membrane of memory cells, it fluidizes or disorganizes the membrane, which leads to
changes in the physical and biochemical characteristics. Chronic exposure alters the
composition of the membrane as wells as its rigidity. Studies have found that lipid
molecules, in the membranes of brain cells of animals fed alcohol for a prolonged time
can confer alcohol resistance to brain cell membranes of animals that have not. Brain
lipids are modified but also brain proteins, demonstrating that brain chemistry is modified
by chronic exposure to alcohol (McGraw-Hill Encyclopedia, ‘97).
Alcohol Wernicke-Korsakoff Syndrome, one of the long-term effects of alcohol
consumption, is two disorders that can occur simultaneously or independently.
Wernicke's disease involves damage to multiple nerve cells in the brain and spinal cord, it
also includes symptoms caused by alcohol withdrawal. Korsakoff syndrome involves the
impairment of memory and intellect skills, which include learning. Korsakoff psychosis
involves damage to areas of the brain.
Alcohol Withdrawal Syndrome is a set of symptoms that occur when one
suddenly stops drinking after long term consumption of alcohol. The spectrum of alcohol
withdrawal symptoms ranges from such minor symptoms as insomnia and tremulousness
to severe complications such as withdrawal seizures and delirium tremens. In alcohol
withdrawal syndromes, the brain maintains neurochemical balance through inhibitory and
excitatory neurotransmitters. The main inhibitory neurotransmitter is gammaaminobutyric acid (GABA), One of the major excitatory neurotransmitters is glutamate,
which acts through the N-methyl-D-aspartate (NMDA) neuroreceptor. Alcohol inhibits
NMDA neuroreceptors, and chronic alcohol exposure results in up-regulation of these
receptors. An abrupt stop to alcohol exposure results in brain hyperexcitability, because
receptors previously inhibited by alcohol are no longer inhibited (Bayard, ’04).
Alcohol has different affects based on different genetic makeup. Gender has an
affect on the impaired. Women get impaired quicker then males while drinking the same
amount of alcohol. This may happen because of the water levels in both are not equal.
However, women are able to eliminate more alcohol per unit in an hour than men.
Women are also more susceptible to alcohol’s long term effects. Heavy drinkers are
mostly found in men. Only 2% of all women are alcoholics while 9% of all men are
alcoholics (Mumenthaler, 1999).
The gene that affects the addiction to alcohol is ADH. The alcohol dehydrogenate
gene is mainly meant to metabolize alcohol. Without it people are much more susceptible
to becoming addicted to alcohol. Different types of the fruit fly Drosophila Melanogaster
have this gene as well (Winberg, 2004). Studies by Heberlein have shown Drosophila
has increases of speed, loss of motor control, and when exposed to the ethanol gas for
over 20 minutes, they become sedated (Heberlein, 2000).
Short-term memory is formed when an outside stimulus is presented to the brain;
the CREB gene, located in the nucleus, is then activated which induces transcription
factors. The RNA enters the nucleus of the cells and copies the CREB gene, and the
messenger-RNA then leaves the cell and executes the genes instructions. The cyclic
AMP cycle is initiated and sodium is flushed into the brain. This sodium causes the
spaces between synapses in the brain to temporarily shrink, allowing for the formation of
short-term memory. Short-term memory in drosophila can last up to 24 hours. Memories
are thought to be due to lasting synaptic modifications within the brain. The rutabega
adenylyl cyclase, an enzyme that is expressed in the Drosophila brain and mediates
synaptic plasticity is needed in cells of the mushroom bodies for a component of
olfactory short-term memory (Zars 2000).
The cyclic AMP systems plays a crucial role in olfactory learning in drosophila;
calcium ions enter vertebrate cells through a glutamate receptor know as the NMDA
receptor. This neural stimulation leads to a movement of calcium ions into the cells
leading to activation of adenyl cyclase and a rise in cyclic AMP levels. Cyclic AMP then
activates protein kinase A, which causes a phosphorylation cascade leading to the
induction of genes involved in learning (Davis 1995).
Protein kinase A, also known as the cyclic-AMP dependent protein kinase, is an
enzyme that supplies proteins with phosphate groups. This protein kinase A activity is
regulated by fluctuating cyclic-AMO within cells and is ultimately responsible for all the
cellular responses due to the cyclic-AMP second messenger system (Bowen 2003).
Literature Review
Moore, 1998, exposed drosophila to ethanol and found that drosophila displayed
behaviors that are similar to behaviors of rodents and humans who are intoxicated,
including lack of balance, and impaired motor coordination. An inebriometer, a 4 ft long
glass column containing multiple oblique mesh is baffles in which ethanol vapor is
circulated, was used to measure ethanol- induced loss of posture control. An inebriometer
is a tube in which the flies are placed in towards the top and are meant to “stick” to the
level, as the intoxication increases, the fall layer and layer. To begin a trial flies are
introduced into the top of the inebriometer. With time, flies lose their ability to stand on
the baffles and gradually tumble downward. As they fall out of the bottom of the
inebriometer, a fraction collector is used to gather them at 3 min intervals, at which point
they are counted. The elution profile of wild-type control flies follows a normal
distribution Cheapdate a mutant drosophila with enhanced sensitivity to ethanol was
used. It was found that it is an allele of the memory mutant amnesiasic, a fly that has been
postulated to encode a nueropeptide that activates the cAMP pathway. It was found that
cheapdate can be reversed by treatment with stimulators that increase cAMP levels or
PKA activity.
Miller tested human’s short-term memory a week after alcohol consumption, in
which one group of people were consuming alcohol throughout a week and another group
was given a placebo. After testing short-term memory he found that alcohol treated
subjects displayed significantly poorer short-term memory compared to those without
alcohol consumption, demonstrating that short-term memory is affected by alcohol
consumption.
Farr allowed mice to become dependednt on alcohol by adding it into their food
and exposing them to it constantly since birth. He then abruptly stopped giving them
alcohol for a three-week withdrawl period. The learning and long-term memory tests
included olfactory footshock avoidance and shuttlebox active avoidance. His findings
included permanent defects of neurochemical changes in the brain, demonstrating
permanent, irreversible, effects on the brain.
Sullivan, 2000, developed a scale of 0-7 on how Drosophila reacts to ethanol and
cocaine. When Drosophila have normal behavior they are a 0 on the scale; a 1 is intense
nearly continuous grooming; a 2 is a loss of negative geotaxis and flight; a 3 is slow
locomotion in a circular motion; 4: sideways or backward locomotion; 5: hyperkinetic
behaviors including wing bussing, erratic activity with flies often bouncing off the walls;
6: sever body tremors, no locomotion; 7: death.
Heberlein, 2004, demonstrated the effects of ethanol concentration on the sedation
times of Drosophila. There were three different concentrations used, 56% alcohol, 50%
alcohol, and 43% alcohol. The higher concentrations caused more drosophila to become
intoxicated
LaFerriere, 2008, studied the correlation between ethanol sensitivity and learning
mechanisms in Drosophila. The learning phenotypes of 10 new strains of drosophila were
examined, and 5 were found to have increased ethanol sensitivity. They trained the files
using place and olfactory memory; individual flies were trained in a heat box to avoid
part of a small chamber by associating one half with a temperature that they avoid, 41o,
with a contrasting temperature of 24o, In this test, one-half of the chamber is heated to the
same temperature as that used for conditioning while the other half of the chamber is kept
at 24°, a temperature that flies normally prefer. After being trained for 20 minutes the
memory was tested for 3 minutes, directly following the training, once the high
temperature was removed. In the other, more classic test, olfactory conditioning paired
one of two odorants (4-methylcyclohexanol and octanol) with electric shock (100 V).
Memory tests were performed 3 min after training, where changed olfactory preferences
were tested in a t-maze. Besides finding new memory mutant flies, they found no
significant correlation between ethanol sensitivity and place memory in the 10 lines
tested. She found that the 3-54 mutant memory remains elevated relative to wild-type
performance even 3 hr after training. Furthermore, the slopes of the memory decay
curves in the two lines are roughly parallel.
Purpose
Therefore the purpose of this experiment is to demonstrate the effects of alcohol
exposure on short-term memory in drosophila.
Hypothesis
The null hypothesis states that the ethanol exposure will have no effect on the
short term memory of Drosophila melanogaster. The alternate hypothesis states the flies
exposed to ethanol before training will have impaired memory and the flies exposed to
alcohol after training will not have damaged memory.
Methodology
Drosophila Melanogaster were the model organism in this study. This model
organism was divided into 5 groups. The first group was a control in which no drosophila
will be exposed to alcohol. The next group was the organism that was exposed to 200proof Ethanol before training and then a group that was exposed after training.
The flies were placed in a training container lined with a 1-milimeter flexible
circuit board, All-Flex, which gave the flies a tingling sensation in their feet. For 1
minute the flies were shocked while simultaneously being pumped an odor 3-octonal
which has been found to not be preferable in flies. The shock intervals consisted of 1.5
square pulses with 1.5 seconds of rest, which was repeated for 60 seconds. They were
times to complete the training protocol. The scent was administered using an air
pump while the shock was supplied with a Grass Telefactor S48 Square Pulse Stimulator.
The flies were then returned to the vial with the supplemented food.
5 hours after training, which is sufficient short term memory for flies, the flies
were tested by being lowered in an elevator into two scent chambers between two scents,
the familiar scent 3-octanol, which they were previously shocked with, as well as a new
unfamiliar scent 4-methycyclihexanol, which is found to not be preferable in flies. The
flies that moved away from the 3-octanol scent, which they were previously shocked
with, were said to have formed short term memory, since they had linked the odor with
the foot shocking, while those that remained in the middle or went to the old scent did
not form short-term memory, since they could not make the connection.
Alcohol was exposed to Drosophila through respiration. Drosophila were placed
in a testing chamber which was connected through a tube to a beaker. 200-proof ethanol
was placed in the beaker and an air pump was connected in to the beaker. The alcohol
was evaporated by an air pump allowing a steady stream of ethanol to follow into the
chamber where the drosophila are held. The drosophila were exposed for 5 minutes either
before or after training.
Discussion: The purpose of this experiment is to demonstrate the effects of
alcohol exposure on short term memory in drosophila. The data supported part of the
alternate hypothesis, the files exposed to ethanol before training will have impaired
memory, because the control group demonstrated short-term memory formation but
the flies exposed to ethanol before and after training did not. The independent
variables in the graph were the control, alcohol before and the alcohol after. The
dependent variable was the performance index. The Performance Index is a way to
measure if learning occurred. Since there were 3 groups, learning occurred if the
performance index was above 33%. The three groups for the most part had a variety
of results. The control group had a high performance index where learning occurred.
The two groups given alcohol were below the 33% because they did not use olfactory
memory, or their memory was impaired from the alcohol, to connect the scent with
the shocking. There was variation between the control and the two alcohol groups.
The control was higher, while the two alcohol groups were very similar in results,
with the alcohol after being slightly higher. The flies were counted as those who
avoided the scent, including the flies that stayed in the middle, and the flies that went
towards the scent. In the individual trials, the flies had choice between the familiar
scent that they were shocked with, and a new unfamiliar scent. Ideally, the files
should have avoided the familiar scent and gone to the unfamiliar scent. For the most
part, the flies choose a scent within the 4 minutes they were given, but in every trial
there were also flies that stayed in the middle and did not decide. Although the flies
did avoid the familiar scent, they did not pick the other scent. It is difficult to decide
what to do with the flies in the middle because there are a variety of things that could
affect them. They could still be drunk, they could be dead, or they could just not
know what to pick. But they could also be avoiding the familiar scent. The results did
not make sense because the performance index of the flies with alcohol was, with
alcohol before, 7.8%, and alcohol after, 10.8%. They both seemed to defy the
expected outcome because even if the flies had just guessed, the percentages would
have been higher. Another data analysis was made excluding the middle flies. All of
the groups increased. The control went from 50.5% to 75%, the alcohol before
changed to 35% and the alcohol after became 37%. These results made more sense
because the variables with the alcohol were higher and more likely. The alcohol
before and after were still both very close, showing that they had a similar effect on
the brain.
Conclusion- The data supports the alternate hypothesis because the control group
demonstrated short term memory formation, while the alcohol groups did not.
Limitations: Limitations included possible human errors. Also more trials would’ve
been helpful for the flies given alcohol. Future studies would include looking at the
long term effects of alcohol on the brain.
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