Involvement of GABAA receptors containing α6 subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging

Abstract

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABA_A receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABA_A receptors, containing the α₆ subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.

Keywords: 2PE uncaging; GABAA receptor; RuBi-GABA; antisecretory factor; cerebellar granule cells; patch-clamp technique.

FIGURE 1

The image illustrates different uncaging points on soma (1), axon initial segment (AIS) (2) and neurite (3) of a cerebellar granule cell that has been processed to reveal biocytin conjugate with CF®640R during patch‐clamp recording

FIGURE 2

Typical chloride current measurements evoked by the uncaging of 10‐μM RuBi‐GABA (750 nm, 100 ms, 30 mW, −80 mV) at about 2 μm from the soma, demonstrating the current (pA) versus time (s). Firstly, 10‐μM RuBi‐GABA was uncaged (a), then, the neurons were incubated for 3 min with 1‐μM AF‐16 (b) and, finally, the current was recorded after the photorelease of 10‐μM RuBi‐GABA in combination with 1‐μM AF‐16 (c)

FIGURE 3

Example of chloride current traces evoked by the uncaging of 10‐μM RuBi‐GABA (750 nm, 100 ms, 30 mW, −80 mV) on soma (a), axon initial segment (AIS) (b) and neurite (c) on the same cell: (A) current trace after the photorelease of 10‐μM RuBi‐GABA, (B) incubation for 3 min with 1‐μM AF‐16 and (C) current measured after the uncaging of 10‐μM RuBi‐GABA in combination with 1‐μM AF‐16

FIGURE 4

Example of chloride current traces evoked by uncaging 10‐μM RuBi‐GABA (750 nm, 100 ms, 30 mW, −80 mV) on the soma of the same cell. (a) and (c): (A) Current trace recorded after the photorelease of 10‐μM RuBi‐ GABA, (B) incubation for 3 min with 1‐μM AF‐16 and (C) current measured after the uncaging of 10‐μM RuBi‐GABA in combination with 1‐μM AF‐16 and, only for trace c, 1 mM furosemide. (b): (A) Current trace recorded after the photorelease of 10‐μM RuBi‐GABA, (B) wash‐out with external solution for 1 min and (C) current measured after the uncaging of 10‐μM RuBi‐GABA in combination with 1 mM furosemide