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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