Purpose The crayfish neuromuscular junction (NMJ) has been used as a model for central nervous system synaptic transmission due to its dual innervation by excitatory (glutamate) and inhibitory (GABA) neurons to each muscle fiber. Local application of 5HT has demonstrated the ability to enhanced excitatory junction potential (EJP) amplitudes with excitatory nerve stimulation and can produce spontaneous, repetitive depolarizations of the post-synaptic membrane. The original purpose of this study was to (1) examine the phenomena of histeresis associated with automatic EJPs. Secondarily, we observed changes in responsiveness to 5HT near the end of local crayfish season, which has been studied and demonstrated by others.
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 and averaged over 10 seconds following the start of stimulus onset. The limb was bathed in concentrations of 5HT ranging from 0.5 to 500 μM. EJPs were obtained within 30 seconds of application and then again at 5 minutes. Recordings were made of automatic EJPs to characterize frequency and amplitude changes. When assessing for histeresis, the exchange of the bath was done by aspirating out the bath and replacing it with the next lower concentration.
Results 1. Histeresis was assessable only in one instance, and was not present in a drop from a threshold level of 1.1 μM to subthreshold 1.05 μM. 2. A significant change in responsiveness to 5HT was demonstrated over the last two weeks of locally available crayfish, in comparison to results 2-6 weeks prior. Threshold for automatic EJPs was seen at 1.0 to 1.3 μM from July 9 to Aug 19, all bathing in 5-HT approximately 2-3 minutes before onset. Thereafter, a transition of increasing threshold and onset to automaticity occurred until 8-25-04, at which point no automatic EJP spiking occurred in the presence of 5HT in the range of 1-500 μM.
Conclusion (1) Histeresis was not seen in the study when decreasing from a threshold level of 1.1 μM 5HT to 1.05 μM 5HT. (2) The species of crayfish studied demonstrated a change in responsiveness to 5HT in regard to ability to elicit automatic EJPs in the range of 1.0 to 500 μM, which correlates with known seasonal variability in 5HT sensitivity.
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