The aim of this experiment was to explore the effects of the neuropeptide FMRFamide (FMRFa) on the crayfish neuromuscular junction (CNJ). In previous experiments, DF2, a FMRFa-like peptide, has been shown to increase neurotransmitter (NT) release at the CNJ. We hypothesized that, like DF2, FMRFa would also potentiate the release of NT and thereby increase excitatory postsynaptic potential (EPSP) amplitude. We also hypothesized that IBMX, a phosphodiesterase inhibitor, would be needed to potentiate the excitatory effects of FMRFa, which it has been shown to do with DF2. To test these hypotheses, we exposed the crayfish to both a high and low Ca2+ Ringer’s solution and to various treated solutions. The first treated solution contained just FMRFa, the second IBMX, and the third both FMRFa and IBMX. We then measured NT release by EPSP amplitudes, recorded from postsynaptic muscle cells via intracellular recording. Our data showed a trend that in both low and high Ca2+Ringer’s solution, FMRFa did increase neurotransmitter release, supporting our first hypothesis. Our second hypothesis was rejected, for our data showed that in a FMRFa and IBMX treated solution average EPSP amplitude decreased.
Phe-Met-Arg-Phe-NH2, or FMRFa, is a neuropeptide from a family of FMRFa-related peptides called FaRPs. DF2, belonging to this family of FMRFa-like peptides, has been shown to increase neurotransmitter (NT) release at the crayfish neuromuscular junctions (CNJ) (Skerret et al2005). Concerning the excitatory effects of DF2, Friedrich et al (1998) suggest that protein kinase C is involved, while Badhwar et al (2006) posit involvement of protein kinase A and G. Additionally, Noronha and Mercier (1995) claim that Ca2+-calmodulin dependant protein kinases are necessary for this excitatory effect of DF2. It has also been suggested that nitric oxide (NO), a retrograde messenger, is involved in FMRFa’s ability to increase NT release. Rõszer et al (2006) suggest that FMRFa is a substrate source of NOS synthase in the gastropod nervous system, because it enhances NO liberation. Our experiment explored the relationship between FMRFa and DF2, a relationship that has not fully been addressed. Our experiment specifically tested to see if FMRFa would increase NT release at the CNJ, as DF2 has previously been shown to do.
We hypothesized that FMRFa would increases NT release at theProcambarus clarkii neuromuscular junction, increasing EPSP amplitude. We also hypothesized that IBMX, a phosphodiesterase inhibitor that slows the break down of cAMP and cGMP, would be needed to potentiate the excitatory effects of FMRFa (Badhwar et al 2006). To test our hypotheses we exposed the superficial dorsal extensor muscles in the tail of the crayfish to various solutions and measured their EPSPs. The first solution contained a low Ca2+ Ringer’s solution, while the test solution was treated with FMRFa, then IBMX, followed by a combination of both. We repeated this process in a high Ca2+ solution to see if calcium was needed to potentiate the effects of FMRFa.
While our data was statistically insignificant, our results did indicate a trend towards slightly increased EPSP amplitude in low Ca2+ Ringer’s solution with FMRFa present. Our data was, however, statistically significant in the high Ca2+, showing an increase in EPSP amplitude. Overall, our data supported our first hypothesis. Our second hypothesis that IBMX was needed to potentiate the effects of FMRFa was rejected. Our data showed that IBMX decreased the effects of FMRFa, suggesting that the excitatory effects of IBMX on DF2 are not analogous to those on FMRFa.
MATERIALS AND METHODS
The low Ca2+ Ringer’s saline solution was comprised of KCl 5.4 mM, NaCl 196 mM, MgCl2.6H2O 7.1 mM, Na Hepes Buffer 10 mM, and CaCl2.2H2O 6.0 mM, adjusted to a pH of 7.4. This solution was changed every 15 minutes. We then diluted the same solution with a 100 mM stock solution of FMRFa, to a concentration of 100 μM. To make our second test solution, we diluted the original Ringer’s solution with a 100 mM stock solution of IBMX to a concentration of 100μM. This was done with a micropipettor. The third test solution was the Ringer’s solution diluted down to a 100μM concentration of FMRFa stock solution and a 100μM concentration of IBMX stock solution. We then prepared the first and third test solution with a high Ca2+ Ringer’s solution comprised of KCl 5.4 mM, NaCl 196 mM, MgCl26H2O 2.6 mM, Sodium Hepes Buffer 10 mM, and CaCl22H2O 13.5 mM with pH of 7.4.
The preparations were changed daily, and exposed to the solution for at least ten minutes before recording. We exposed our preparation to the FMRFa solution and IBMX solution on different days. Additionally, the preparation was always exposed to the FMRFa + IBMX soltuion directly after it was exposed to either the FMRFa or IBMX solution.
The crayfish were purchased from Carolina Biological Supply Company (North Carolina, U.S.A.). The crayfish were kept in an ice bath before dissection. We then cut alongside the tail’s ventral surface and removed the swimmeretes, intestines, and abdominal muscles, leaving behind the dorsal superficial extensor muscle. We pinned the crayfish by two pins in a 200ml dissection dish, with silicone elastomer. We then poured the various saline solutions over the crayfish into the dish.
We used two electrodes, a recording microelectrode and nerve-stimulating suction electrode. We pulled apart the 1.2mm microelectrode with a PUL-1 micropippette puller. We then filled it with 3 M KCl, rinsed it in a saline solution, and placed it on the micromanipulator. The resistance of the microelectrodes were consistently measured to be >20 MΩ.
Nerve Stimulation and Data Recording
The suction electrode was connected to a stimulator, which stimulated the captured pre-synaptic nerve. It was stimulated with single pulses at a frequency of 0.5 Hz and at the lowest voltage possible to measure an EPSP. Using the micromanipulator we then placed the electrode in the muscle most lateral to the captured nerve to record the signals in the post-synaptic muscle cell. These signals were passed through an amplifier and recorded with the computing program Scope.
To see if FMRFa increased NT release we compared the EPSP amplitudes recorded from the crayfish tail while in a high Ca2+ Ringer’s solution and while in a solution diluted with FMRFa. The differences in the mean EPSP amplitudes were statistically significant (p =0.003), showing us that FMRFa increased the EPSP amplitude. FMRFa increased the EPSP amplitude on average by 6.6 mV, which supports our hypothesis regarding the excitatory effects of FMRFa (Figure 1).
Figure 1. Average EPSP amplitude before and after exposure to FMRFa. High Ca2+: n=5, error bar indicates standard error of 2.5. High Ca2+ + FMRFa: n=4, error bar indicates standard error of 3.3. The p value is 0.003.
We then tested the effects of FMRFa in a low Ca2+ solution. The average EPSP amplitude recorded in the control solution was 12.6 mV, while it increased to an average of 13.5 mV when FMRFa was added. These results, while showing a trend that FMRFa increases NT release, were statistically insignificant (p=0.052). To test the hypothesis that IBMX would potentiate the effects of FMRF, we added both IBMX and FMRFa to the control solution. Our results show that IBMX decreased the effects of FMRFa on average by 3.8 mV, causing us to reject our hypothesis (Figure 2). This result was statistically significant (p2+solution.
Figure 2. Average EPSP amplitude before and after exposure to FMRF and IBMX. Low Ca2+: n=18, error bar indicates standard error of 1. Low Ca2++ FMRFa: n=13, error bar indicates standard error of 0.8. Low Ca2++FMRFa+IBMX: n=14, error bar indicates standard error of 1.1. p2+ to low Ca2++FMRFa+IBMX, and when comparing low Ca2++FMRFa to low Ca2++FMRFa+IBMX. p=0.052 when comparing low Ca2+ to low Ca2++FMRFa.
After our second hypothesis was rejected, we tested to see if IBMX would have the same negative effects if applied alone without FMRFa. To test this we compared the EPSP amplitude in a low Ca2+ solution to the EPSP amplitude in a low Ca2++IBMX solution. While the p value was 0.552, we saw that IBMX increased the EPSP amplitude on average by 2mV, n=4.
Our first hypothesis was that FMRFa would increase EPSP amplitude. Overall, our data supported a trend that FMRFa increases NT release (Figure 2). While we did see this trend, our data revealed unclear results. For example, in low Ca2+ our data was statistically insignificant, while it was significant in high Ca2+. In addition to this, our results varied from day to day, showing how biological variability affected our experiment. Our second hypothesis was that IBMX would potentiate the effects of FMRFa. Our data rejected this hypothesis, suggesting that IBMX in combination with FMRFa does not enhance FMRFa’s effects, but rather reverses them (Figure 2).
Our experiment raises questions of the role of IBMX on NT release. Previous studies suggested that IBMX enhanced the effects of DF2(Badhwar et al. 2006; Morley et al. 2009). Despite the fact that DF2 is a member of the FMRFa family, our data suggests that IBMX does not have the same effects on FMRFa that it does on DF2. This observation is important, for the target receptor of FMRFa is still unclear. Therefore, there is room for further study as to whether or not FMRFa truly increases NT release, and if so by what mechanism it achieves this. Rőszer et al (2006) suggest that FMRFa is a substrate source of nitric oxide (NO), which could suggest that NO acts as a signaling molecule, increasing NT release. To test this question, L-NAME, an inhibitor of NO synthase, could be used to see whether the effects of FMRFa could be halted or reversed, suggesting that NO is involved in its excitatory effects.
Our data was the most unreliable in the high Ca2+ because the high concentration triggered action potentials that jeopardized our data collection. Our data, however, was statistically significant in high Ca2+, showing the largest difference between the EPSP amplitude of the control and that of the FMRFa solution (Fig. 1). Further testing is required to see how calcium affects the possible excitatory effects of FMRFa. Further tests could include using a calcium chelator, such as EGTA, to see if the observed excitatory response of FMRFa would be reversed by lower concentrations of Ca2+. This could suggest a possible target receptor for FMRFa.
In testing for the direct relationship between the EPSP amplitude of low Ca2+ and low Ca2++ IBMX, our data, although statistically insignificant (p=.552), showed that IBMX alone increased the EPSP amplitude by 2mV. This data was not as informative as our other data, because there were only four recordings taken to test the effect of IBMX. If these results were supported by future tests, they would suggest that FMRFa reverses the excitatory effects of IBMX. This would give us valuable information regarding the mechanism by which FMRFa increases NT release.
We thank Clark Lindgren, our professor, Sue Kolbe, our lab assistant, and our mentors Grace Hazeltine and Molly Wingfield for assisting us in the lab and helping us interpret our experiment.
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