Habituation-Like Decrease of Acetylcholine-Induced Inward Current in Helix Command Neurons: Role of Microtubule Motor Proteins

Abstract

The role of kinesin and dynein microtubule-associated molecular motors in the cellular mechanism of depression of acetylcholine-induced inward chloride current (ACh-current) was examined in command neurons of land snails (Helix lucorum) in response to repeated applications of ACh to neuronal soma. This pharmacological stimulation imitated the protocol of tactile stimulation evoking behavioural habituation of the defensive reaction. In this system, a dynein inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, 50 µM) decreased the ACh-current depression rate. Kinesin Eg5 inhibitors (Eg5 inhibitor III, 10 µM and Eg5 inhibitor V, trans-24, 15 µM) reduced the degree of current depression, and Eg5 inhibitor V also reduced the initial rate of depression. The results of electrophysiological experiments in combination with mathematical modelling provided evidence of the participation of dyneins and kinesin Eg5 proteins in the radial transport of acetylcholine receptors in command neurons of H. lucorum in the cellular analogue of habituation. Furthermore, these results suggest that the reciprocal interaction between dynein and kinesin proteins located on the same vesicle can lead to reverse their usual direction of transport (dyneins-in exocytosis and kinesin Eg5-in endocytosis).

Fig. 1

An example of depression of the ACh-induced inward current in an LPa3 neuron in a cellular analogue of habituation. a ACh-currents to rhythmical applications of acetylcholine are presented. Numeric ciphers indicate the ordinal numbers of ACh application in the series. Holding potential is −75 mV. b Scheme of stimulation protocol in the experiment. T test ACh applications before and after the series

Fig. 2

Influence of the dynein inhibitor EHNA on the dynamics of ACh-current depression in a cellular analogue of habituation. Overall results of all experiments are presented. Horizontally—time (min); vertically—ACh-current amplitude (mean ± SEM)  % relative to ACh-current value in response to the first application of ACh in series. Squares EHNA (50 µM), circles no drugs, Helix saline, control. Vertical lines SEM, *P < 0.05—level of significance of the differences of the amplitudes of the ACh-current in the experiment and control (Mann–Whitney test)

Fig. 3

Influence of Eg5-I–III, a kinesin Eg5 inhibitor, on the dynamics of ACh-current depression in a cellular analogue of habituation. The overall results of all experiments are presented. Horizontally—time (min); vertically—ACh-current amplitude (mean ± SEM) % relative to ACh—current value in response to the first application of ACh in series. Squares Eg5-I–III (10 µM), circles DMSO (0.1 % control). The remaining symbols are as in Fig. 2

Fig. 4

Influence of Eg5-I–V, an inhibitor of kinesin Eg5, on the dynamics of the neuronal ACh-current depression in a cellular analogue of habituation. Squares Eg5-I–V (15 µM), circles DMSO (0.1 % control). The remaining symbols are as in Fig. 2

Fig. 5

Mathematically modelled curves for the number of membrane acetylcholine receptors, which are assumed to determine the depression of acetylcholine-induced inward current. 1—control [without pharmacological influence a and after actions of 0.1 % DMSO (b)]; 2,3,4—after actions of inhibitors, EHNA (2a), Eg5-I–V (2b, 3b), Eg5-I–III (4b): 2b—if the coefficients, describing retrograde transport of receptors to the membrane are reduced; 3b, 4b—if both the coefficients modelling retrograde transport and the coefficients modelling direct receptor transport are reduced