MRS5474, in contrast to a canonical A1receptor agonist, did not affect hippocampal excitatory inputs to excitatory neurons in control conditions
We first assessed the mechanism of action of MRS5474 by evaluating its ability to affect excitatory synaptic transmission at the CA1 area of the rat hippocampus, a brain area mostly affected by seizures and where the inhibitory action of adenosine A1R agonists is well known (Sebastião et al., 1990). To our surprise, MRS5474, at a concentration (120 nM) nearly 40 times higher than its affinity for A1R (Carlin et al., 2017), was virtually devoid of effect upon excitatory postsynaptic currents (EPSCs) recorded by patch clamp from CA1 pyramidal cells in the whole cell configuration (Figure1 ). At the end of the perfusion with MRS5474 (120nM), the EPSC peak amplitude was 106±8.0% of the pre-drug value (n=6, P>0.05, Figure 1A ). In two out of the six cells, there was even a tendency to an increase (Figure 1A-B ) rather than the expected decrease. Holding current and membrane resistance were also not affected by MRS5474 (120 nM) (Holding current: 2.6±4.8 pA, n=6, P>0.05; membrane resistance: 100±3.5% of pre-dug values, n=6, P>0.05). The absence of effect of MRS5474 was still evident in experiments where the membrane impermeant sodium channel blocker, QX-314 (5 mM), was added to the intracellular solution of the patch electrode (peak amplitude at end of application 105±6.1% of pre-drug values, n=5, P=0.4353, Figure 1C ). In contrast to the absence of effect of MRS5474, the canonical A1R agonist, CCPA (30 nM) clearly inhibited the EPSC peak amplitude within 3–5 minutes after its application, being the maximal effect attained after about 20 minutes (70 ± 5.2% inhibition, n=4, P<0.05, Figure1D ).
Next, we evaluated if MRS5474 could inhibit excitatory synaptic transmission under less restrictive recording conditions – field EPSP recordings from hippocampal slices, and in the same rodent where the antiseizure action was detected, i.e., the mouse. We tested concentrations ranging from 120 nM up to 500 nM of MRS5474 and again, no significant effect (P>0.05, n=7) was detected (% change at the end of application of 120 nM, 250 nM and 500 nM: with inhibition of 11 ± 10.2%, -2.7 ± 9.9% & -9.3 ± 10.6% respectively, Figure2A ). Under similar experimental conditions, the canonical A1R agonist, CPA (30nM), caused the expected inhibition of fEPSPs (-50 ± 9.7% inhibition, n=2, P<0.05, Figure 2B).