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  • To apply the recently developed GPR ligands

    2022-01-19

    To apply the recently developed GPR35 ligands to CNS disorders, it is necessary to understand whether GPR35 is expressed by any identified cell types in the ML324 and whether activation or blockade of this receptor has any consequence on the activity of the neuronal circuitry. To this end, the present study was designed to test the hypothesis that GPR35s are expressed by hippocampal neurons and that activation of these receptors leads to suppression of neuronal excitability. To test this hypothesis, we used an antibody anti-GPR35 to determine whether GPR35s are present in hippocampal neurons and examined the effects of GPR35-selective pharmacological agents on the frequency of fast current transients (CTs) that correspond to action potentials recorded from stratum radiatum interneurons (SRIs) under cell-attached configuration in rat hippocampal slices. Results presented here lend support to the test hypothesis and indicate that GPR35s are potential molecular targets for controlling neuronal activity in the brain.
    Materials and methods
    Results
    Discussion The present study provides the first direct evidence that GPR35s are expressed by neurons in the CA1 field of the rat hippocampus and that activation of these receptors suppresses the rate of firing of CA1 SRIs. Although a previous study suggested that the gene encoding GPR35 can contribute to the brachydactyly mental retardation syndrome [17], the potential relevance of functional GPR35s to regulation of brain functions has been largely disregarded. Recently, Berlinguer-Palmini et al. [18] reported that the mRNA encoding GPR35 was present in cultured cortical astrocytes and that GPR35 activation suppressed excitatory transmission in hippocampal slices. Here, we demonstrate that an antibody that specifically detects rat GPR35s in transiently transfected HEK 293 T-cells is able to label cells in rat hippocampal slices. The GPR35-immunopositive cells visualized in the rat hippocampal slices studied here were neurons, as they were NeuN-immunopositive and GFAP-immunonegative. Based on their location, most of the GPR35-labeled, NeuN-positive cells were interneurons in various layers of the hippocampus. It is possible that the expression level of GPR35 in astrocytes in the hippocampal slices studied here was below the detection threshold of the immunofluorescence assay used here and/or that expression of GPR35 by astrocytes is brain-region-specific. Several rapid screening assays have been developed to assess the pharmacology of GPR35 ligands [2], [11], [19]. Most assays involve expression of structurally modified GPR35 and/or co-expression of promiscuous G-protein α subunits in cell lines. In high-throughput systems, expression of a chimeric G protein α subunit switches the preference of GPR35 from Gi/o or Gs to Gq, and, consequently, evokes a rise in the level of inositol phosphate and intracellular Ca2+, each serving as a readout for receptor activation [2], [7], [20], [21]. These assays, which are efficient, convenient, and allow rapid screening of large numbers of molecules led to the identification of a battery of pharmacological tools essential for identification of functional GPR35s [12], [13], [21], [22]. In the present study, we used two experimental protocols, each having advantages and limitations, to evaluate the effects of GPR35 ligands on neuronal firing. In one protocol, hippocampal slices were kept in either ligand-free ACSF or in ACSF containing GPR35 agonists for >2h before recordings were initiated. This allowed us to test the effect of various ligands under equilibrium conditions. Using this method, the EC50s of agonists to suppress the frequency of SRI firing were estimated to be <1μM for zaprinast, and >10μM for pamoic acid (Table 1). This is in agreement with the known apparent potency for these ligands to activate rat GPR35s. The limitation of this approach is that the end-point (frequency of firing) was assessed from different set of slices for each treatment condition, thus creating more variability in the data. In the second protocol, the effects of ligands were assessed in the same cells from which control data had been obtained, thereby minimizing the variability in the data. However, as the effects were monitored within 25–30min of exposure of the slices to the agonists, it was not possible to attain complete equilibrium conditions. Yet, the results of this second set of experiments provided a rough estimate of the EC50 for inhibition of the frequency of SRI firing. Using this non-equilibrium condition, EC50s were estimated to be ∼1μM for zaprinast, ∼50μM for dicumarol, <1mM for pamoic acid, and ∼3μM for amlexanox (Table 1). Clearly, the EC50s of agonists were higher in non-equilibrium than in equilibrium conditions. However, the order of potency for suppression of the frequency of SRI firing was comparable between the two protocols: zaprinast=amlexanox>dicumarol>pamoic acid. This rank order of potency is consistent with that reported for GPR35 activation using other biochemical measurements (Table 2). The fact that the concentrations of GPR35 agonists in our assay were higher than those found in other rapid screening assays simply suggests that the native receptor under physiological conditions may have different sensitivity compared to receptors expressed in artificial cell systems. Such variation in potency for agonists is also evident in biochemical measurements using different assays (Table 2).