Influence of Membrane Receptor Lateral Diffusion on the Short-Term Depression of Acetylcholine-Induced Current in Helix Neurons

Abstract

We have studied how various drugs increasing the rate of nicotinic acetylcholine receptors (nAChRs) lateral diffusion affect the depression of ACh-induced current in land snail Helix lucorum neurons responsible for defensive behavior. The acetylcholine (ACh) iontophoretic application protocol imitated the behavioral habituation protocol for the intact animal. We found that the drugs decreasing cholesterol level in cell membranes as methyl-β-cyclodextrin 1 mM and Ro 48-8071 2 µM, and polyclonal antibodies to actin-binding proteins as spectrin 5 µg/ml and merlin 2.5 µg/ml have changed the dynamic of ACh-current depression. The nAChRs lateral diffusion coefficient was obtained by fluorescence recovery after photobleaching. A curve fitting model specially created for analysis of short-term choline sensitivity depression in snail neurons helped us evaluate separately the contribution of nAChRs lateral diffusion, their endocytosis and exocytosis to observed effects during electrophysiological experiments. Taken together, we hypothesize that nAChRs lateral diffusion plays an important role in the cellular correlate of habituation in land snail Helix lucorum neurons.

Keywords: Acetylcholine receptors; Acetylcholine-induced current, Habituation-like short-term depression; Command Helix neurons; Lateral diffusion; Methyl-β-cyclodextrin; Ro 48-8071; Spectrin, merlin.

Fig. 1

Changes in ACh peak current amplitude in the cellular correlate of habituation. a An example of ACh-current depression during repeated ACh applications to soma of the LPa3 neuron. Blue control, red after MβCD (1 mM) application. Holding potential −75 mV. b The schematic representation of the experimental stimulation protocol

Fig. 2

Effect of MβCD on the dynamics of ACh-current depression in the cellular correlate of habituation. The overall results of the experiments are presented. X axis time, minutes; Y axis ACh peak current amplitude (mean ± SEM), as a percentage of the neuronal response on the first neurotransmitter application. Squares MβCD (1 mM), circles no drugs, Helix saline, control. Vertical lines standard error of mean, red circles P < 0.05 (the confidence level of the differences between experimental and control ACh peak current amplitudes, Mann–Whitney U test)

Fig. 3

Effect of Ro 48-8071 on the dynamic of ACh-current depression in the cellular correlate of habituation. The overall results of the experiments are presented. X axis time, minutes; Y axis ACh peak current amplitude (mean ± SEM), as a percentage of the neuronal response on the first neurotransmitter application. Squares Ro 48-8071 (2 μM). Circles control. The remaining labels are the same as in Fig. 2

Fig. 4

Effect of antibodies to spectrin (Ab-Spectrin) on the dynamics of ACh-current depression in the cellular correlate of habituation. Squares Ab-Spectrin (5 µg/ml), circles control. The remaining labels are the same as in Fig. 2

Fig. 5

Effect of antibodies to merlin (Ab-Merlin) on the dynamics of ACh-current depression in the cellular correlate of habituation. Squares Ab-Merlin (2.5 µg/ml), circles control. The remaining labels are the same as in Fig. 2

Fig. 6

An example of FRAP experiment on Helix neurons expressed nAChRs labeled with fluorescent (CF405S) α-Bungarotoxin. CF^TM405S is a blue fluorescent dye with an absorption peak wavelength that nearly coincides with the 405-nm blue diode laser line. a The confocal images of the cell membrane of Helix neuron during FRAP experiment. A bleached region is marked by a yellow circle. The background signal was collected from the extracellular space. The unbleached region was screened for photobleaching correction. b This plot illustrates the average of normalized intensity of recovery after photobleaching (n = 7) as a function of time with error bars indicating standard errors of the mean

Fig. 7

Fitted curves describing the changes in normalized nAChRs number, which are assumed to determine the depression of ACh-induced inward current after MβCD exposure. X axis time, minutes; Y axis the normalized number of membrane acetylcholine receptors calculated as a percentage of the number of membrane acetylcholine receptors before the first neurotransmitter application. 1 control; 2–4 after MβCD: a as the result of 1.5-fold (Curve 2), 2-fold (Curve 3), and 3-fold (Curve 4) raise of receptor diffusion coefficient only; b as a result of 3-fold decrease in constant of REndo rate and 3.8-fold (Curve 2), 3.5-fold (Curve 3), and 3.8-fold (Curve 4) decrease in constants of RExo rate, respectively (without changing the receptor diffusion coefficient); c as a result of changes some model parameters as compared with control (increase of diffusion coefficient by 2 times and decrease of endocytosis and exocytosis rates by 3 and 3.8 times, respectively)

Fig. 8

Fitted curves describing the changes in normalized nAChRs number, which are assumed to determine the depression of ACh-induced inward current after application of antibodies to spectrin and merlin. 1 control (without pharmacological influence), 2,3 after actions antibodies to spectrin (2) and merlin (3). The remaining labels are the same as in Fig. 7