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).