Biochemical assays have provided evidence that prolonged exp

Biochemical assays have provided evidence that prolonged exposure to GPR35 agonists can induce GPR35 internalization [2], [4], [11], [13]. Our finding that neuronal excitability decreased regardless of whether the slices were superfused for a few minutes or incubated for a few hours with GPR35 agonists strongly suggests that receptor activation rather than desensitization is the major determinant of the effects of agonists under the present experimental conditions. It is possible that the association of G-protein minimized the magnitude of desensitization of GPR35 in the neurons, whereas the non-inclusion of G-proteins in the biochemical assays increased the magnitude of desensitization of GPR35 in cell lines. The fact that multiple ligands suppressed the frequency of SRI firing with the rank order of potency compatible with their agonistic actions on GPR35s and that their effect was antagonized by the GPR35 antagonist ML145 supports the contention that functional GPR35s are present in the hippocampal neurocircuitry including SRIs. The partial antagonistic effect of ML145 can be attributed to the fact that this compound is a weak antagonist at the rat GPR35 [13], and currently no other potent GPR35 antagonist is available for the rat species. Although pamoic Dexmedetomidine HCl is known to inhibit deoxyribose phosphate lyase and DNA polymerase activities at the range of concentrations used in our study [23], it is unlikely that these effects led to a reduction in the action potential frequency in the CA1 SRI. Other actions of the test ligands that could have led to suppression of SRI firing were ruled out. For example, blockade of NMDA receptors can suppress the frequency of CTs recorded from neurons in the Mg2+-free model used here. However, zaprinast (10μM) was shown to be devoid of any antagonistic action of NMDA receptors, as it had no significant effect on NMDA-evoked currents in the SRIs. It is unlikely that the GPR35 agonist-induced suppression of CT frequency originated from a reduction in the tonic α7 nAChR activity in the SRI because the α7 nAChR antagonist methyllycaconitine itself is least effective in decreasing the action potential frequency in these neurons [16]. In addition, direct inhibition of phosphodiesterase by the ligands tested here is unlikely to contribute to the suppression of SRI firing because sildenafil (see Fig. 9) and cromoglycate (otherwise known as cromolyn; see Fig. 6A), known inhibitors of phosphodiesterase [24], [25], failed to suppress the frequency of firing of the interneurons. The observations that sildenafil decreased the inhibitory effect of zaprinast suggest that sildenafil may be acting as an antagonist at the GPR35 in our experimental conditions. The exact mechanism by which GPR35 activation suppresses the rate of firing of SRIs remains to be determined. However, it is noteworthy that hyperpolarization-activated cation currents (Ih), which are activated by elevated intracellular levels of cAMP and are known to contribute to the generation of spontaneous firing, are present in hippocampal SRIs [26], [27]. Thus, considering that GPR35 activation has been shown to inhibit adenylate cyclase via Gi/o pathway and cause a decrease in intracellular cAMP levels in multiple systems [5], [8], it is conceivable that agonist-induced activation of GPR35s in SRIs reduces intracellular cAMP levels and, consequently, leads to suppression of Ih currents, which, in turn, decreases the rate of firing of the interneurons (Fig. 10). The implications of the presence of GPR35s on hippocampal interneurons are not yet fully understood. However, the finding that superfusion of the hippocampal slices with the GPR antagonist ML145 caused a significant increase on the spontaneous firing of CA1 SRIs supports the concept that GPR35s are tonically active on these neurons and regulate either their firing threshold or firing pattern. A recent study [28] demonstrated that intracerebroventricular injection of amlexanox prevented ischemia-induced loss of the nuclear protein prothymosin alpha from CA1 pyramidal neurons. This effect has been explained by the inhibitory action of amlexanox on S100A13, a Ca2+-binding cargo protein [28]. However, based on the report that amlexanox is a potent GPR35 agonist [10] and on our present observation that this ligand suppresses the firing frequency of CA1 SRIs, it is tempting to speculate that GPR35 plays an important role in ischemia-related mechanisms in the hippocampus. In fact, a recent study found that GPR35 is overexpressed in cardiomyocyte cell membranes after hypoxia [29].