Purpose The crayfish neuromuscular junction (NMJ) has been used as a study for central nervous system synaptic transmission due to its dual innervation by excitatory and inhibitory neurons to each muscle fiber. Likewise, the NMJs in the walking limb utilize glutamate and GABA as excitatory and inhibitory transmitters, respectively. Many neurotransmitters have been studied at this site, including serotonin (5HT). Application of 5HT locally enhances excitatory junction potential (EJP) amplitudes with excitatory nerve stimulation and can produce spontaneous, repetitive depolarizations of the post-synaptic membrane. This study characterizes automatic EJPs in the presence of 5HT at various concentrations and assesses concentration of threshold for automaticity.
Methods The excitatory nerve innervating the dactylopodite opener muscle was isolated in first or second walking limbs of locally obtained crayfish (Oncogenes rusticus). Standard intracellular electrophysiological recording techniques were used to study amplitudes of EJPs evoked at 30 Hz. Control EJPs were recorded in Van Herreveld's solution (Van H) and averaged over 10 seconds following stimulus onset. The limb was bathed in 5HT concentrations ranging 0.5 to 500 μM. EJPs were obtained within 30 seconds of application and then again at 5 minutes. Recording were made of automatic EJPs to characterize frequency and amplitude changes.
Results Threshold of automaticity occurred at 1.0 to 1.3 μM with onset of automaticity occurring from 2-3 min post-application of 5-HT. Automaticity at 5 μM had onset times of 1-2 min, 10 μM at 30 s, 25 μM at 0-30 s, and 50 μM occurring immediately post-application. There were significant trends toward increasing EJP amplitude and frequency which tended to stabilize 30-60 s post-application. In all instances, post-5HT washout resolved automatic EJPs.
Conclusion Automatic EJPs in post-synaptic muscle membrane without application of electrical stimulus in the presence of 5-HT was observed. There was an inversely graded response in time to onset with threshold to automaticity in the range of 1.0 to 1.3 μM 5-HT. Increasing frequency and amplitude tended to be seen within the first 30-60 s of automaticity onset, which then stabilized. Concentrations of 5-HT from 0.5 to 0.9 μM were assessed in three preparations without observing automatic EJPs.