As a neuroscience concentrator at Grinnell, you use methods and theories spanning disciplines from biochemistry to psychology to investigate the function of the nervous system.
Endocannabinoids are a widely studied family of molecules that act as neurotransmitters and influence synaptic plasticity. Endocannabinoids have been extensively studied in the frog and lizard neuromuscular junction. However, this study assessed the effects of arachidonylcyclopropylamide (ACPA), an agonist to the CB1 cannabinoid receptor, in the crayfish neuromuscular juction. We found that the addition of ACPA decreased the excitatory post synaptic response. We cannot conclusively determine if an endocannabinoid analogue utilizes the CB1 receptor pathway in the crayfish neuromuscular junction.
Cannabinoids are a group of molecules that function as neurotransmitters and include ∆9-tetrahydro-cannabinol, or THC, the active ingredient in marijuana. These molecules have motor as well as psychoactive effects that occur upon their interaction with receptors on nerve cell membranes. There are two subtypes of cannabinoid receptor, CB1 and CB2, and activation of either receptor leads to activation of a G-protein, then decreases in levels of protein phosphorylation and intracellular cyclic AMP (cAMP). The endogenous cannabinoids, or endocannabinoids, which activate these receptors are the fatty acids 2-arachidonylglycerol (2-AG) and anandamide (Sánchez-Pastor et al. 2007).
At the frog neuromuscular junction, anandamide has been shown to activate CB1 receptors and block the enzyme adenylate cyclase, which converts ATP into cAMP (Van der Kloot 1994). Administration of cannabinoids at the frog neuromuscular junction also decreases MEPP frequency through the activation of presynaptic CB1 receptors. These receptors interact with Gi/o proteins and activation seems to lead to the blockage of N-type Ca2+ channels (Sánchez-Pastor et al 2007).
Also in the frog neuromuscular junction, two CB1 receptor agonists, WIN and ACEA, have opposite effects on quantal acetylcholine (ACh) release. Introduction of WIN and the CB1 antagonist AM 251 nullified WIN’s depression of ACh release. Introduction of ACEA yielded an increase in ACh quantal release, and this increase was not negated when AM 251 was introduced. Silveira et al. (2010) discovered that ACEA targets the vanilloid receptor (TRPV2) and is blocked by capsazepine, the TRPV1 antagonist.
Cannabinoids have also been studied in the lizard neuromuscular junction. Newman et al. (2007) examined the presence and location of CB1receptors via fluorescence microscopy. They also questioned whether M3muscarinic ACh receptors inhibit neurotransmitter release via endocannabinoids. By utilizing multiple combinations of agonists and antagonists, the researchers also determined whether Ca2+ influx was actively being inhibited in the nitric oxide-dependent system. The researchers discovered that Anolis carolinensis do in fact have CB1receptors that are localized on the presynaptic nerve terminal. When muscarine and AM 281, a CB1 antagonist, were applied they determined that the depression of EPP was due to cannabinoid receptors. They also discovered that endocannabinoids cause a decrease in transient Ca2+that accounts the later depression of synaptic transmission. In conclusion, Newman et al. (2007) discovered that through the M3 muscarinic receptor mediated system, muscle cells synthesize endocannabinoids (2-AG) to bind to CB1 receptors on the nerve cell.
Our study hoped to fill the void bridging endocannabinoids in the crayfish NMJ, which have shown to play a part in synaptic plasticity. Ultimately we hope to draw comparisons with other previously studied model organisms. By drawing these comparisons, we can further test if the specific mechanisms published in the frog and lizard also are expressed in the crayfish. The goal(s) of this study are/were to test whether the crayfish NMJ is sensitive to activation of CB1 receptors, and to determine whether the response seen, due to cannabinoid addition, affects the CB1receptor pathway.
We hypothesized that, similar to the lizard and frog NMJ, the crayfish would show analogous effects when the CB1 agonist, ACPA, was introduced – a decrease in excitatory post synaptic potential (EPSP). We also hypothesized that any effect seen due to the addition to endocannabinoids would be caused by augmentation of the CB1 receptor.
MATERIALS AND METHODS
Experimental preparation and solutions
Crayfish were first immersed in crushed ice for approximately 10 min. Next, we removed the tail and isolated the tail extensor muscle. This muscle and the attached exoskeleton were pinned down in a Sylgard®-coated dish. We covered the preparation in approximately 40 ml of fresh physiological saline solution (5.4 mM KCl, 196 mM NaCl, 13.5 mM CaCl2•H2O, 2.6 mM MgCl2•H2O, 10 mM HEPES).
Arachidonylcyclopropylamide (ACPA) was obtained in Tocrisolve®, a soy oil and water emulsion. It was diluted in physiological saline before being added to the crayfish preparation at an approximate final concentration of 10 µM. ACPA was purchased from Tocris Cookson (Ellisville, MO, USA).
End-plate potentials were evoked by stimulating the motor nerve axon at 1-10 V for 5 ms, at 0.2 Hz. We used a glass micropipette filled with 3 M KCl and with resistance between 5 and 15 MΩ to measure EPPs and recorded data using a MacLab data acquisition system (AD Instruments, Colorado Springs, CO, USA).
For our first experiment, we recorded from a number of separate, randomly chosen muscle cells both before and after treatment with ACPA. The average EPP amplitudes in each condition were compared using a student’s t-test (two-sample assuming equal variance).
For our second experiment, we recorded from several separate, randomly chosen muscle cells before the addition of ACPA. After drug treatment, EPPs were measured in one cell every 30 sec for 2 min and then every 1 min for 18 min. Mean EPP amplitudes before and after treatment were compared as above.
For our third experiment, we recorded from one muscle cell both before and after ACPA treatment. EPPs were averaged over 8 stimuli for recording. We recorded two averages before treatment, and several averages at a number of time intervals after treatment. In Figure 2, each bar after ACPA addition is the mean of four recordings. Mean values for consecutive pairs of averages were compared using a student’s t-test (two-sample assuming equal variance).
In order to determine whether cannabinoids have an effect at the crayfish neuromuscular junction, we performed electrophysiology of the tail extensor muscle. We recorded EPSPs while stimulating the motor nerve at low frequency, both with and without the presence of the CB1 receptor agonist ACPA.
We first compared the average value of five EPSPs recorded from randomly selected muscle cells before and after ACPA treatment (Figure 1). This recording did not show a significant change in EPSPs due to the addition of ACPA.
Figure 1 (top panel). Mean EPSP amplitude. Each bar represents the mean of six EPSPs, and all data was recorded from one crayfish muscle preparation.
Second, we performed a time course experiment in which we recorded from one cell both before and after ACPA treatment (Figure 2). We found a significant difference between the mean EPSP before ACPA addition (35.60 mV) and the mean EPSP after addition (15.66 mV) (p = 0.024). ACPA decreased the EPSP.
Figure 2 (center panel). Time course of the effect of ACPA on the crayfish neuromuscular junction. ACPA was added at 1 min. Each point represents one recorded EPSP (n=1). After ACPA addition, EPSPs were recorded every 30 s for 2 min and then every 1 min for 18 min.
Third, we performed an experiment in which we recorded EPSPs averaged over eight nerve stimuli, also in one cell (Figure 3). We found a significant difference between the mean EPSPs recorded before treatment with ACPA and those recorded from 0 to 2.5 minutes, 3.5 to 6 minutes, and 7 to 10 minutes after treatment (p values = 0.002, 0.0003, and 2 × 10-5, respectively). In this experiment, as well, ACPA decreased the EPSP.
Figure 3 (bottom panel). Effect of ACPA on average EPSP amplitude (± SEM). Each bar after ACPA addition represents the mean of four average recordings, while the bar before ACPA represents the mean of two. * indicates p < 0.05 compared to EPSP before ACPA. (n=1)
Experiment 1 did not provide any statistically significant results (Figure 1). This lack of significance is primarily since there is a large variation in EPSP amplitude across different muscle cells. To counteract this difficulty, it might help to survey cells that were similar distances from the nerve that was being stimulated. We could have also used a larger sample size, which might have allowed us to find a statistically significant result. Additionally, we did not allow any time to pass for our first measurement after we added ACPA, which may have affected the results of our experiment. Due to these limitations, the results of this experiment neither validate nor disprove our hypothesis.
In the second trial, the addition of ACPA showed a statistically significant effect of decreasing the EPSP 43.9% (Figure 2). With the exception of a few points, the time course showed a consistent decrease as each successive measurement was taken. This was the first indication that the addition of a CB1 receptor agonist would decrease the EPSP in the crayfish NMJ. This result supports our hypothesis.
In our third experiment, we found a significant effect of ACPA (Figure 3). The agonist led to EPSP depression which increased over time. Although the methodology of this experiment was somewhat different from the previous one, they are similar enough that their results reinforce each other’s validity. Therefore, this experiment supports the conclusions we drew from our second experiment. Overall, we find that the CB1 receptor agonist ACPA leads to EPSP depression which increases over time. We hypothesize that this consistent enhancement in EPSP depression may be caused by continuing activation of some receptor which responds to ACPA. This may be the CB1 receptor or an analogue of the CB1 receptor.
One of the major limitations in our study was that all three trials employed different methodologies. This inherent lack of continuity calls into question the replication and significance of our results. The opportunity to replicate each of our experiments would have greatly increased the confidence with which our results may be interpreted. However, because our second two experiments are methodologically similar, they can be viewed as confirming each other’s results in the absence of exact replication.
Another major limitation is that we were not able to probe the question of whether an endocannabinoid analogue affects a CB1 receptor in the crayfish NMJ, utilizing both an agonist and an antagonist. If blocking any present CB1 receptors with an antagonist eliminated the EPSP depression in response to agonist treatment, we would be able to conclude that depression is due to a CB1 receptor. Although we confirmed our hypothesis that adding a CB1 agonist does depress EPSP amplitude, we cannot definitively determine whether the effect of endocannabinoids influences the CB1 receptor specifically in the crayfish NMJ.
Future studies teasing apart the relationships of endocannabinoids in the crayfish NMJ would help to extend the results of this study. Further replication of our experiments is needed to strengthen our conclusions. Moreover, it would be helpful to determine the involvement of the CB1receptor using an antagonist. Additionally, further studies could use fluorescence microscopy to spatially locate CB1 receptors on the synapse, for example using fluorescently tagged CB1 antibodies, or add various combinations of agonists and antagonists to the NMJ. In conclusion, we determined that addition of an endocannabinoid analogue, ACPA, decreased the EPSP at the crayfish NMJ.
We would like to thank Clark Lindgren, our professor, for his direction in the classroom, providing necessary background knowledge, and in the lab with troubleshooting our study. Without Professor Lindgren's assistance this experiment would not have been possible. Additionally, we would like to thank the Grinnell College Biology Department for funding our study and therefore giving us the opportunity to pursue neurobiological inquiry.
Newman, Zachary; Malik, Priya; Wu, Tse-Yu; Ochoa, Christopher; Watsa, Nayantara; Lindgren, Clark A. (2007). Endocannabinoids mediate muscarine-induced synaptic depression at the vertebrate neuromuscular junction. European Journal of Neuroscience 25:1619-1630.
Sanchez-Pastor, E.; Trujillo, X.; Huerta, M; Andrade, F. (2007). Effects of cannabinoids on synaptic transmission in the frog neuromuscular junction. Journal of Pharmacology and Experimental Therapeutics321:439-445.
Silveira, P.E.; Silveira, N.A.; Morini, V.C.; Kushmerick, C.; Naves, L.A. (2010). Opposing effects of cannabinoids and vanilloids on evoked quantal release at the frog neuromuscular junction. Neuroscience letters473:97-101.
Van der Kloot, W. (1994) Anandamide, a naturally-occurring agonist of the cannabinoid receptor, block adenylate cyclase at the frog neuromuscular junction. Brain Research 649:181-184.
View this article as PDF: Morley, Shriver, and Zhang
This experiment looked at the role of nitric oxide in the mechanism by which the peptide DF2 increases neurotransmitter release at the crayfish neuromuscular junction. We hypothesized that if DF2 increases neurotransmitter release through a pathway involving nitric oxide as a retrograde signal, then when L-NAME, an inhibitor of nitric oxide synthesis, was applied, DF2 will not increase neurotransmitter release. To test this we exposed crayfish superficial dorsal extensor muscles to DF2, which was amplified with IBMX. We then submerged the preparation into saline solution that contained DF2, IBMX, and L-NAME. Neurotransmitter release was measured by the amplitude of EPSP traces recorded from the postsynaptic muscle cells via intracellular recording. Our data shows an increase in neurotransmitter release after exposure to DF2 and IBMX, as well as a decrease in neurotransmitter release after the addition of L-NAME, a trend that supports our hypothesis. Further testing is needed to draw conclusions about our hypothesis.
DRNFLRFamide, AspArgAsnPheLeuArgPhe-NH2, (DF2), is a peptide that enhances synaptic transmission at neuromuscular junctions. However, the mechanism by which DF2 accomplishes this is still unclear (Friedrich et al. 1998; Badhwar et al 2006). Badhwar et al (2006) suggest that protein kinase A (PKA) and protein kinase G (PKG) are involved in the pathway by which DF2 increases EPSP amplitude. Friedrich et al. (1998) claim that protein kinase C (PKC) is needed in this process. Calcium-calmodulin dependent protein kinases, such as CaMKII, also have apparent roles in mediating the effects of DF2 in the presynaptic terminal (Noronha and Mercier 1995). According to Badhwar et al. (2006), it is plausible that nitric oxide is required in the DF2 signaling pathway as a retrograde signal because of its small molecular size, high membrane permeability, and the presence of membrane-bound guanylyl cyclase in crustaceans. Badhwar et al. (2006) hypothesized that nitric oxide increases cGMP levels, which is involved in the DF2 pathway, via soluble guanylyl cyclase. Our study explores the role of nitric oxide as a possible retrograde messenger in the mechanism by which DF2 increases neurotransmitter release.
The antagonist L-NAME was utilized in this study because of its ability to block nitric oxide production. L-NAME blocks nitric oxide production because the structure is similar to that of the amino acid L-Arginine. This similarity in structure allows L-NAME to act like L-Arginine and bind with nitric oxide synthase, the nitric oxide production enzyme, to stop nitric oxide production. We hypothesized that if DF2 increases neurotransmitter release through a pathway involving nitric oxide as a retrograde signal, then when L-NAME is applied to inhibit nitric oxide synthesis, there will be a decrease in neurotransmitter release.
To test this hypothesis we exposed crayfish superficial dorsal extensor muscles to L-NAME to block nitric oxide production. We expected DF2 to increase the neurotransmitter release, and that L-NAME would cause a decrease in neurotransmitter release. Our results show that DF2, when enhanced with IBMX, causes an increase in neurotransmitter release. Our results also suggest that exposure to L-NAME prevents this increase in neurotransmitter release.
MATERIALS AND METHODS
We used crayfish (Procambarus clarkii), which were stored at 20 °C, and put on ice before the experiment. We cut the tail off a crayfish and then cut along the sides of the tail, cutting as close to the ventral part of the tail as possible. The cephalothorax and surrounding tissues and muscle cells were removed so that only the exoskeleton of the dorsal surface and the superficial extensor muscles along the dorsal surface remained. The tail was placed into the dissection dish, pinned, and covered with 25mL of crayfish saline solution.
Ringer solutions were prepared with three different chemicals by dilution with a low calcium crayfish ringer solution (Table 1). This control solution had a pH of 7.4 and consisted of 5.4mM KCl, 200.7mM NaCl, 12.3mM MgCl2 • 6H2O, 5mM Sodium Hepes Buffer and 5mM CaCl2 • 2H2O. We used a lower calcium solution to inhibit cells from triggering action potentials.
Table 1. Composition of Saline Solutions
|Saline||DF2 (mM)||IBMX (mM)||L-NAME (mM)|
The first preparation was exposed to the control saline followed by saline A. The second preparation was exposed to the control, then saline B, followed by saline C. Each preparation was submerged into 25mL of saline solution, which was replaced with new solution every 15-30 minutes. Tests involving DF2 were completed without changing the saline solution until a new chemical was added because we had such a small amount of DF2 to work with. These tests were completed as quickly as possible to protect against fatigue and cell death.
We used two kinds of electrodes, suction electrodes for nerve stimulation and microelectrodes for recording. Both electrodes were fitted to manipulators and their respective reference electrodes were submerged in the saline solution. The suction electrode was put into an electrode holder that allowed saline to be drawn through the holder by a syringe. Recording electrodes were pulled from glass capillary tubes with 1.2mm diameter, filled with 3M KCl, and inserted into an electrode holder, which was also filled with 3M KCl. Recording electrodes had resistances ranging from 4MΩ to more than 10MΩ.
Nerve Stimulation and Recording
The suction electrode was attached to a stimulator, which stimulated the pre-synaptic nerve that was sucked into the electrode. The nerve was stimulated with single pulses at a frequency of 0.5Hz, and at the lowest voltage possible to measure an EPSP.
Using a microscope and micromanipulator, we inserted the microelectrode into a muscle cell in the same segment and on the same side as the nerve that was being stimulated. The recording microelectrode recorded the signals in the post-synaptic muscle cells. The signals passed through an amplifier and the membrane potentials and EPSP traces were viewed using the Scope program.
We tested the involvement of nitric oxide in the DF2 signaling pathway, through which DF2 increases EPSP amplitude. First, we exposed one preparation to DF2 to see if DF2 affects EPSP amplitude. We then applied DF2 and IBMX, followed by L-NAME to another preparation to see if blocking nitric oxide production alters the effect of DF2 on EPSP amplitude.
We compared the EPSP amplitudes recorded before and after we applied DF2. Our data shows that DF2 does not increase EPSP amplitude when applied alone. DF2 decreased the EPSP amplitude by 28.4% (Figure 1).
Figure 1. Average EPSP Amplitude Before and After Exposure to DF2
The average control amplitude was 8.4 mV (n=4) and the average DF2amplitude was 6.4mV (n=3). After exposure to DF2, the amplitude dropped 24% (p>.05, student t-Test). Error bars indiciate standard error of 2.72 for the contrl and 0.66 for DF2.
We compared the change in EPSP amplitude before and after we applied both DF2 and IBMX on another crayfish preparation. The average EPSP amplitude for the control was 5.61 mV, and the average EPSP amplitude for DF2 and IBMX was 6.58 mV. The average EPSP amplitude increased 17.3%, which demonstrates the effect of DF2 and IBMX on EPSP amplitude. We then compared the EPSP amplitudes before and after L-NAME was added. Our results showed that the average EPSP amplitude after the sample was exposed to L-NAME was 3.22 mV, 51% lower than the average amplitude for DF2 and IBMX (Figure 2). The average amplitude for L-NAME was also 42.5% lower than the average for the control (Figure 2).
Figure 2. Average EPSP Amplitudes.
The average EPSP amplitude for DF2 and IBMX trials (n=5) is higher than the control average (n=4, p>.05), and the L-NAME trials (n=5) have a lower average than both DF2 and control trials (p>.05 for both comparisons). Error bars show standard errors, control: 3.06, DF2 and IBMX: 2.21, and L-NAME: 1.51.
In our experiment, we observed that DF2 did not increase neurotransmitter release when applied alone. Although we do not have much data to support this result, these results are contrary to the results presented by Badhwar et al. (2006). This data could suggest a flaw in our assumptions that DF2 should increase EPSP amplitude per Badhwar et al (2006), but more data is needed for such a conclusion to be drawn from these results.
As suggested by Badhwar et al (2006), when a significant increase in EPSP amplitude was not observed after adding DF2, we added IBMX, which should have enhanced the effects of DF2. When DF2 and IBMX were both added to the preparation the EPSP amplitude increased, which indicates an increase in neurotransmitter release. Although we do not have a significant amount of data to draw conclusions, this result is consistent with the findings of Badhwar et al (2006).
Our data shows that the EPSP decreases when L-NAME is added to a preparation that contains DF2. This preliminary data suggests that nitric oxide is involved in the pathway by which DF2 increases neurotransmitter release. Further study is needed to support or refute this trend. This data would support our hypothesis that nitric oxide is involved in the mechanism by which DF2 increases EPSP, as the necessary nitric oxide should be unavailable because L-NAME blocks the synthesis (Newman et al, 2007).
The average EPSP amplitude for L-NAME was also lower than the average control amplitude. This could be due to a few reasons. One possibility is that inhibiting nitric oxide production with L-NAME may affected mechanisms other than the DF2 mechanism. Our design did not allow for targeted application of chemicals, so it is possible that the application of L-NAME affected more than just the effect of DF2 and IBMX. The other very plausible option is that the amplitudes were generally lower because the cells were fatigued and beginning to die. Although saline solutions were replaced at relatively regular intervals, it is possible after about one and a half hours, the crayfish muscle cells were beginning to die.
Future research on this topic would include more trials using L-NAME and DF2 to see if more data supports our hypothesis the way our preliminary data suggests. If our hypothesis is supported, the next step would be to test the role of nitric oxide as a retrograde signal using carboxy-PTIO. This would also support the suggestion that nitric oxide is involved in a retrograde signaling pathway that Badwar et al. (2006) present in their paper. More tests should be done to measure nitric oxide levels, and to test if the DF2 receptors are located on the post-synaptic membrane using fluorescent markers.
We thank Clark Lindgren, our professor, Sue Kolbe and Abby Griffith, our lab assistants, and Adhiti Kannan, our mentor, for their assistance with this project. We would also like to thank our willing crayfish friends, Joe, Bob, Sally, and Snuffleupagus.
Badhwar, A., Weston, A., Murray, J., & Mercier, A. J. (2006). A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide. Peptides, 27, 1281-90.
Friedrich, R., Molnar, G. F., Schiebe, M., & Mercier, A. J. (1998). Protein Kinase C Is Required for Long-Lasting Synaptic Enhancement by the Neuropeptide DRNFLRFamide in Crayfish. The Journal of Neurophysiology, 79(2), 1127-1131.
Newman, Z., Malik, P., Wu, T., Ochoa, C., Watsa, N., & Lindgren, C. (2007). Endocannabinoids mediate muscarine-induced synaptic depression at the vertebrate neuromuscular junction. European Journal of Neuroscience, 25, 1619-30.
Noronha, K.F. and Mercier, A.J. (1995). A role for calcium/calmodulin-dependent protein kinase in mediating synaptic modulation by a neuropeptide. Brain Research, 673 (1), 70.
Skerrett, M., Peaire, A., Quigley, P., Mercier, A.J. (1994). Physiological Effects of Two FMRFamide-Related Peptides from the CrayfishProcambarus Clarkii. The Journal of Experimental Biology, 198, 109–116.