Evidence from Paired-Pulse Experiments Suggest that Endocannabinoids Do Not Inhibit Neurotransmitter Release by Reducing Calcium Influx

Abstract

Endocannabinoids are involved in retrograde messaging in neurons. Recent studies have found evidence of endocannabinoids and CB1 receptors at the neuromuscular junction of vertebrates as well as in some invertebrates. We attempted to explain how endocannabinoids affect neurons by examining the influence of CB1 receptors on paired-pulse facilitation at the neuromuscular junction, Procambarus clarkii. We measured excitatory postsynaptic potentials (EPSP) at the neuromuscular junction using standard electrophysiological techniques with twin-pulse stimulation and applied the endocannabinoid receptor agonist, ACPA and antagonist AM 251. We then compared the ratio of twin-pulse amplitudes of the EPSPs during the application of agonist and antagonist to our baseline to determine whether there had been any change to inracellular Ca²⁺ in the presynaptic cell. We hypothesized that the ratios of EPSP2 to EPSP1 would vary between the conditions with ACPA, AM 251 and the baseline. This would have indicated that Ca²⁺ concentrations were different after the application of an agonist. Although our data suggests that ACPA does have an effect on inhibiting the release of neurotransmitters in the crayfish neuromuscular junction, our evidence shows that ACPA does not mediate synaptic inhibition of neurotransmitters through inhibition of calcium ion channels. This research has the potential to redefine the current studies of endocannabinoids receptors in invertebrate and augment our understanding of their role in the nervous system.