not-yet-known not-yet-known not-yet-known unknown MRS5474 induced a dose-dependent potentiation of GABA-evoked currents in oocytes micro-transplanted with human hippocampal membranes. The enhanced A3R immunoreactivity in hippocampal samples from patients with epilepsy, suggesting seizure-induced overexpression of A3R, together with the finding that MRS5474 can activate A3R to affect GABA transporters prompted us to evaluate the action of MRS5474 upon GABAergic transmission in human tissue. Thus, we used the technique of micro-transplantation of Xenopus oocytes with human tissue, which allows assessing receptor-mediated neurotransmitter responses and the use of human tissue taken from autopsies or surgeries even in quantities that would not allow other kinds of functional studies (Miledi et al., 2002; Palma et al., 2003; Ruffolo et al., 2020). We recorded GABA (500µM)-evoked currents from oocytes injected with hippocampal membranes of three epileptic patients (clinical details in Table 3). The amplitude of these currents ranged from 8.0 to 249.0 nA (n = 47; #1–3). Incubation with MRS5474 (5 µM) for 2 hours and 30 minutes (Roseti et al., 2008, 2009), induced a significant enhancement of GABA-evoked responses (P<0.05, 41.0 ± 14.9% as mean of percentage changes; from 47.3 ± 8.9 nA, before incubation to 71.0 ± 16.9 nA, after incubation; n = 15, #1-3; Figure 7A–B). Acute co-application of MRS5474 (5 µM) together with GABA did not exert any effect on GABA-evoked current amplitude (n=8, not shown). As shown in Figure 7B, the effect of MRS5474 was concentration dependent. There was a mild tendency, even though not significant, when using 50 nM of MRS5474 (p=0.055, from 43.0 ± 10.0 nA, before incubation to 46.7 ± 11.0 nA, after incubation; n=8; #1, 2) and higher concentrations produced an increasingly higher potentiation (Figure 7B ). To address the putative involvement of A3R in the effect of MRS5474 upon GABA currents from human hippocampal tissue, we tested its sensitivity to the A3R antagonist, MRS1523 (Li et al., 1998). In this set of experiments, MRS1523 (10 µM) was pre-incubated for 30 minutes and then co-incubated for 2 hours with MRS5474 (1.5 μM). Under such conditions, MRS5474 was virtually devoid of effect (from 45.5 ± 5.4 nA, before incubation with MRS5474 to 46.3 ± 4.6 nA after incubation; #1,2 Table 3) upon GABA currents (Figure 7C), though in the same experiments but in the absence of the A3R antagonist, MRS5474 (1.5μM) caused the usual increase in GABA currents (Figure 7C). Notably, when MRS5474 (5µM) was incubated (2 hours and 30 minutes) with oocytes micro-transplanted with control brain tissues, it did not increase GABA-evoked current (from 40.0 ± 13.6 nA, before incubation to 37.4 ± 9.95 nA after incubation; n = 10; #4; P>0.05). The comparison between the percentage change in GABA currents induced by MRS5474 (5µM) in human epileptic vs non-epileptic tissue shows a highly significant difference (P<0.05, Figure 7D), suggesting that MRS5474 specifically affects epileptic tissue.