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Pioneering Neuroscience, 2002, 3, 41-44
Fluoxetine and hyperforin appear to act like a known glutamate reuptake
inhibitor by increasing EPSP duration in the crayfish neuromuscular junction.
ADAM HOYE, DERRICK MITCHELL, and ALEX TUCKER
Department of Biology, Grinnell College, Grinnell, Iowa
ABSTRACT
Commonly used antidepressants, fluoxetine and hyperforin inhibit serotonin reuptake to treat patients. There is
evidence that one of these selective serotonin reuptake inhibitors (SSRIs), hyperforin, also affects glutamate, another
neurotransmitter (Di Carlo, 2001). Our study was to determine if another SSRI, fluoxetine, had similar effects on
glutamate reuptake inhibition as hyperforin. We first examined the effects of the known glutamate reuptake
inhibitor, aminocaproic acid, in order to establish a framework through which to compare the effects of SSRIs on
glutamate reuptake inhibition. In addition we examined the effects of the SSRIs when they were used in conjunction.
The study was conducted in the crayfish neuromuscular junction due to its simplicity. We compared the duration of
EPSPs under normal and experimental conditions, in order to determine the similarities of the effects on glutamate
reuptake inhibition between the chemicals. It was shown that fluoxetine exhibited the largest increase in duration,
hyperforin showed the smallest increase, and the combination of fluoxetine and hyperforin exhibited an increase in
EPSP duration that was between that of the two SSRIs alone. We contend that fluoxetine had a greater effect on the
reuptake inhibition of glutamate than hyperforin. This questions the safety of using fluoxetine, the main active
ingredient in Prozac due to unsuspected side effects. An unexpected result came when fluoxetine and hyperforin
were used together in that the averaging affect of the two chemicals presents that possibility that combining these
two drugs is safer than using fluoxetine alone, in regards to glutamate reuptake inhibition.
INTRODUCTION
Communication between nerve cells relies upon
neurotransmitters and their ability to send signals
form one neuron to another. Two common
neurotransmitters are serotonin and glutamate.
Serotonin (5-hydroxtryptamine) is a neurotransmitter
involved in a variety of physiological and behavioral
functions ranging from the control of sleep and
wakefulness, feeding, cardiovascular functions,
sexual behavior, spinal regulation of motor functions,
emotional and psychotic behavior, and drug-induced
hallucinatory states (Boyer, W. F., 1994). The
excitatory amino acid, L-glutamate, is a primary
neurotransmitter in the excitatory synaptic pathway
in the central nervous system. Glutamate is the most
abundant neurotransmitter in the brain, and necessary
in the terminals of the spinal cord
(http://www.eb.com:180/bol/topic/?du=119939&sctn
=26#s_top).
When neurotransmitters are released from the
presynaptic neuron into the synaptic cleft, they bind
to receptors on the postsynaptic neuron. These
receptors then relay the signal from the neurotransmitters, called an EPSP, to the rest of the postsynaptic neuron, and release the neurotransmitters back
into the cleft. Pumps on the presynaptic neuron
become activated when an excess of neurotransmitter
© 2002 Grinnell College
is in the cleft after being released by the postsynaptic
receptors, in order to clear the neurotransmitters from
the synaptic cleft. If these reuptake pumps become
disabled, the neurotransmitters will be cleared out of
the cleft by diffusion or degraded by enzymes, but
not before the possibility of binding with
postsynaptic receptors multiple times. When this
happens, the EPSP will have a longer duration, as the
neurotransmitters are left in the cleft for a longer
period of time, during which they can bind and rebind with the postsynaptic receptors.
There are chemicals that selectively inhibit either
serotonin or glutamate reuptake. A chemical that
selectively inhibits glutamate reuptake is 1aminocyclobutane-trans-1,3-dicarboxylic acid
(aminocaproic acid). Alternately, two chemicals that
are selective serotonin reuptake inhibitors (SSRIs)
are hyperforin and fluoxetine. Hyperforin, which is
naturally found in St. John’s Wort, is a popular
antidepressant, as is fluoxetine, which is a
synthetically manufactured compound and the main
active ingredient in Prozac. Research by G. Di Carlo
(2001) states that hyperforin appears to have an effect
on glutamate reuptake pumps. We wanted to
investigate whether the effects of another SSRI,
fluoxetine, on glutamate reuptake pumps are similar
to those of hyperforin. In addition, we first examined
the effects of the known glutamate reuptake inhibitor,
42 A. HOYE, ET AL.
aminocaproic acid, in order to establish a framework
through which to compare the effects of SSRIs on
glutamate reuptake inhibition. As an aside, we
investigated the effects of the two SSRIs in the
presence of one another on glutamate reuptake.
To perform our experiment, we used the crayfish
neuromuscular junction because it was a simple and
accessible way to obtain synaptic recordings. It uses
glutamate as its primary neurotransmitter and was a
simple and fast dissection, which allowed us to
perform multiple experiments in a minimal amount of
time.
In order to determine the effects of glutamate
reuptake inhibition on crayfish, we compared the
duration of EPSPs under normal and experimental
conditions. Because fluoxetine and hyperforin both
inhibit serotonin reuptake, we expected to see similar
effects on glutamate reuptake inhibition as well, by
way of EPSP duration. We also expected to see a
greater increase in inhibition of glutamate reuptake
when hyperforin and fluoxetine were used together,
than when the two chemicals were used
independently. This stemmed from warnings
cautioning consumers to be wary of using the two
drugs at the same time
(http://www.healingwithnutrition.com/products/stjoh
nswort.html).
MATERIALS AND METHODS
Solutions
Standard crayfish saline solution was made of
0.8052g KCl, 24g NaCl, 1.0574g MgCl2.6H2O,
0.3864g NaHCO3, , 4.60 g CaC;2, and 0.7208g
Dextrose. It was necessary that the pH of the saline
solution be 7.4.
1mL of 10mM 1-Aminocyclobutane-trans-1,3dicarboxylic(Aminocaproic) acid was diluted in
standard crayfish saline solution to a total volume of
63mL, and a concentration of 100µM. This solution
was stored in a refrigerator.
We used a 10mg pill of St. John’s Wort, which
was crushed and used to create a 1.48µM hyperforin
solution, which we used in our experiment. The stock
solution was kept in a refrigerator.
One 10mg Prozac tablet was crushed using a
pestle and mortar to make a solution of concentration
of 1.48µM. The stock solution was stored at room
temperature.
Preparation of Crayfish Fast Extensor Muscle Fibers
We obtained a crayfish tail that had been cut
from the thorax of a crayfish. The crayfish had been
desensitized in a tray of ice for no less than 15
minutes. The tail was placed in 50mL of standard
saline solution in a Sylgard-lined preparation dish.
The tail was held and cut longitudinally with
dissection scissors through the shell and flexor
muscles. Connections of the segmental flexor
muscles were cut with laboratory scissors. We then
used small forceps to pull the two halves of the shell
apart, and discarded the ventral portion of the shell.
We then cut the telson off and discarded it. Using the
same small forceps, we removed and discarded the
gut by pulling it away from connective tissues
holding it to the dorsal side of the tail. Finally, the
tail was mounted using 2 pins, driving them through
the shell and into the Sylgard lining of the dish.
Equipment Preparation
We made microelectrodes using a World
Precision Instruments PUL-1 capillary puller. With a
Teflon tipped syringe, we carefully filled our
microelectrodes with 3M KCl. In order to be certain
there were no air bubbles within the microelectrode,
we held the electrode up to a light source and
examined the tip, making sure to remove any bubbles
we saw. We used the same 3M KCl solution to fill a
micromanipulator’s adaptor. The adaptor with
microelectrode was placed in the micromanipulator.
The microelectrode was connected to a
neuroprobe amplifier in order to measure membrane
potentials. A ground probe was placed into the
standard saline solution bath. We used MacLab/4 in
conjunction with Scope v. 3.6.3 software to analyze
our data.
An electrode stimulator connected to a
stimulating source was mounted onto another
micromanipulator.
Measuring Membrane Potentials
We tested the resistance of our microelectrode
setup once the microelectrode was in the solution and
made sure it was in the acceptable range of 5 to 10Ω.
A significant voltage drop (≈30-60mV) that remained
stable indicated to us that the electrode had
penetrated the membrane of a muscle fiber.
Measuring EPSPs
We first positioned the two prongs of the
stimulator straddling the nerve in the caudal margin
of a segment of fast extensor muscle fibers, and then
measured the cell’s membrane potential by
penetrating the muscle fiber with the microelectrode.
Starting with the stimulator on zero volts and on
pulse mode, we monitored the voltage inside the cell
on the computer screen (Scope v. 3.6.3 software).
We slowly increased the voltage of the stimulator
until we observed an abrupt increase in voltage,
which signified an EPSP.
© 2002 Grinnell College, Pioneering Neuroscience, 3 41-44
FLUOXETINE AND HYPERFORIN INCREASE EPSP DURATION 43
Experimental Procedure
Without disturbing the preparation
(microelectrode, stimulator or crayfish), we pipetted
the standard saline solution out of the preparation
dish, making especially sure not to disturb the
microelectrode still within the muscle fiber. We then
replaced the standard crayfish saline solution with
31.5ml of 100µM aminocaproic acid solution.
In order to compare the effects of the chemicals,
we needed to control the variability between the
EPSPs of different cells. We did this by measuring
EPSPs in the same muscle cell under both normal and
chemical conditions. This reduced the variability and
thus made our results more accurate by removing the
difference in EPSPs between cells.
After letting the crayfish soak in the solution for
5 minutes, we stimulated the nerve again using the
same voltage as before. This procedure was repeated
using the hyperforin solution, the fluoxetine solution
and a solution made up of hyperforin and the
fluoxetine. Separate crayfish preparations were used
for each chemical. We took 3 readings of EPSPs
when the crayfish was in standard crayfish saline, and
6 readings when a chemical was present in the
various solutions.
RESULTS
The voltage vs. time graphs of crayfish EPSPs
recorded for each experiment were analyzed by
determining the half-width of each EPSP signal. We
chose to examine the EPSP half-width because
increased EPSP duration is characteristic of
glutamate reuptake inhibition.
The half-widths were determined for each EPSP
measured, and were averaged so that there was only 1
value of a half-width for each condition. We then
calculated the change in average half-widths going
from normal to experimental conditions, and finally
determined the percentage change of these averaged
half-widths (figure 1).
The results obtained from the conditions
containing hyperforin and fluoxetine were referenced
to that of aminocaproic acid. As a known glutamate
reuptake inhibitor, it provided us with a baseline of
what happened to the duration of EPSP signals
during glutamate reuptake inhibition.
Overall, we saw an increase in half-widths when
the crayfish bath changed from normal to
experimental conditions. Fluoxetine exhibited the
largest increase in half-width, which was roughly
45%. Hyperforin showed an increase of 12%, while
the combination of fluoxetine and hyperforin
increased the half-width by 19%. The combination of
fluoxetine and hyperforin percent increase was
© 2002 Grinnell College, Pioneering Neuroscience, 3, 41-44
50
45
40
35
30
25
20
15
10
5
0
Aminocaproic
Hyperforin
Fluoxetine
Fluoxetine & Hyperforin
Solution
Figure 1. Percentage Change of Half-Widths. This chart shows the
percentage change in the half-widths of EPSPs in the crayfish
neuromuscular junction going form standard saline solution to that
which contains the chemicals shown. The percentage changes
shown here are the average of 6 EPSP half-widths in the presence
of the given chemicals divided by 3 EPSP half-widths in standard
saline solution all while in the same muscle fiber. Error bars are
not present because the mean standard error could not be
calculated, as a single crayfish was used for each chemical. A
minimum amount of voltage was used in order to generate an
EPSP. The average voltage needed to generate an EPSP was 48V.
between hyperforin and fluoxetine increase
individually, therefore exhibiting an averaging effect.
A t-test could not be performed for our data
because only one crayfish was used for each
experimental condition, therefore we could not
account for changes in variability due to our
procedure. However, throughout the course of the
experiment, we limited the variability of EPSPs by
examining the same muscle fiber under multiple
conditions. We also compared relative data
(percentage changes in half-width) as opposed to
empirical data (specific to each muscle fiber), which
would be imprecise based on the difference between
crayfish EPSPs, thus limiting variability. However,
we could not account for all variability, and like all
experiments, the accuracy of our experiments could
be improved.
DISCUSSION
As our results stand, they support our hypothesis that
fluoxetine is another SSRI that appears to inhibit
glutamate reuptake. In order to prove this, we first
verified the effects of hyperforin on glutamate
reuptake by comparing its effects with those of a
known glutamate reuptake inhibitor, aminocaproic
acid. This laid the framework to investigate the
effects of fluoxetine, on glutamate reuptake inhibition
as well.
Fluoxetine not only inhibited glutamate reuptake
44 A. HOYE, ET AL.
pumps to the same extent as aminocaproic acid and
hyperforin, it actually exceeded both of them,
contrary to our predictions (figure 1). Because
fluoxetine exhibited such strong results, we can
conclude that fluoxetine acts like hyperforin with
respect to glutamate reuptake inhibition, which
validates our hypothesis.
These results raised interesting questions
concerning the selectivity of reuptake inhibition
between manufactured and naturally occurring
SSRIs. Hyperforin, a naturally occurring SSRI
appears to be more selective in only inhibiting
serotonin reuptake than fluoxetine, which is
synthetically manufactured. This is because
fluoxetine was shown to inhibit glutamate, which is
not a primary function of either SSRI, to a greater
extent than hyperforin (figure 1). This questions the
safety of using fluoxetine, the main active ingredient
in Prozac, because of its lack of selectivity to only
serotonin reuptake pumps, which could lead to
undesirable side effects.
There is also the possibility that there could be a
mechanism of action other than glutamate reuptake
inhibition that the two SSRIs are taking. Although
fluoxetine and hyperforin appear to exhibit the same
characteristics as the known glutamate reuptake
inhibitor aminocaproic acid, we could not be certain
that they function via the same mechanism. Further
experimentation would be needed to conclude the
exact mode of action used by SSRIs to affect
glutamate reuptake inhibition.
For the experiment, we could not obtain pure
hyperforin or fluoxetine (they were in pill form), and
therefore foreign compounds were present in the
solution that could have affected our results. In
addition, monitoring normal EPSPs during bathing
time was not feasible, and therefore any differences
in half-widths were attributed to the effects of the
chemical(s) present and not to experimental
malfunction (e.g. imperfect seal of the cell membrane
around the microelectrode).
A final, unexpected result was that of hyperforin
and fluoxetine used together. The averaging affect of
the two chemicals on percent changes in half-width
of EPSPs raised many questions concerning the
mechanism of interaction between these two
chemicals. This also presents the possibility that
combining these two drugs is safer than using
fluoxetine individually, in regards to glutamate
reuptake inhibition. This supports the warning
provided by healinigwithnutrition.com that states that
using hyperforin and fluoxetine with one another is
more dangerous than taking hyperforin alone.
However, using the two SSRI in conjunction
appeared to have less of an effect on glutamate
reuptake inhibition than the use of fluoxetine by
itself. This may suggest that users of Prozac could
potentially limit side effects caused by glutamate
reuptake inhibition by using St. John’s Wort at the
same time as Prozac, however further
experimentation needs to be performed to validate
this claim.
ACKNOWLEDGEMENTS
We would like to thank Clark Lindgren, Nancy
Rempel-Clower, and Sue Kolbe for providing us with
the materials to perform this experiment, as well as
their support and guidance. We would also like to
thank the lab assistants Babs Lake, Megan
Hagenauer, and Gerald Walther who helped us
through this process.
REFERENCES
Boyer W.F. 1996. The neuropharamacology of
serotonin in the central nervous system. In Feighner
J.P. Selective Serotonin Re-Uptake Inhibitors. John
Wiley & Sons, New York. pg. 1-2.
Di Carlo, G. et. al. 2001. St. John’s wort: Prozac
from the plant kingdom. Trends in pharmacological
science; 22(6): 292-7.
“Nervous system”
<http://www.eb.com:180/bol/topic/?du=119939&sctn
=26#s_top > [Accessed 12 December, 2001]
“St. John’s Wort™: Nutritional Support For
Depression”
<http://www.healingwithnutrition.com/products/stjoh
nswort.html>. [Accessed 19 November, 2001]
© 2002 Grinnell College, Pioneering Neuroscience, 3 41-44