Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Abstract

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and γ-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

Figure 1:. Representative ion currents of oocytes.

Ion currents from oocytes injected with membranes from human synaptic receptors were recorded. AMPA receptors were activated with 100 microM Kainate, and GABA_A receptors with 1 mM GABA. V_H = −80 mV.

Figure 2:. Co-injection of Torpedo and GABAρ1.

Co-injection of filtered membranes (0.1 μm) from the electric organ of Torpedo, rich in acetylcholine receptors, and cDNA coding for the GABAρ1 receptor into the animal pole produced mainly two groups of oocytes based on their responses: one group of oocytes (A) had large responses to acetylcholine (Ach; 1 mM) but no responses to 1 μM GABA (voltage clamped to −80 mV), and oocytes in group (B) had null or low responses to ACh but large responses to GABA. (C) Graph shows mean ± standard error of mean (SEM) of peak current in group 1 (n = 5 oocytes) and group 2 (n = 11 oocytes). One oocyte in group 2 had large GABA and ACh responses suggesting rupture of the nucleus; consequently the distribution of the responses was skewed to low values, as noted by the difference between mean and median of the distribution of membrane current (22 nA vs 6 nA). This result indicates that one of the causes of low-response oocytes is that the membranes are being injected and trapped into the nucleus of the oocyte.

Figure 3:. Polarized insertion of cell membranes in Xenopus oocytes.

GABA and kainate responses of oocytes injected into the animal or vegetal poles, with unfiltered membranes obtained from a non-AD brain (female, 74 years old, postmortem interval 2.8 h) and an AD-brain (female, 74 years old, postmortem interval 4.5 h). Oocytes injected near the animal pole, without targeting the nucleus, gave larger responses than oocytes injected into the vegetal pole, thus allowing the study of tissue samples with very low numbers of receptors. Student's t-test between vegetal and animal poles: ** p < 0.01, *** p < 0.001, Bars indicate mean ± SEM of peak current, n = 5 oocytes each column.