The relatively high concentration of HT used

The relatively high concentration of 5-HT used in this study (100 µM) is consistent with the concentrations used in other published ex vivo studies (Ropert and Guy, 1991, Passani et al., 1994, Shen and Andrade, 1998). In our paradigm 5-HT was focally applied at the surface of the slice via a fast perfusion system. To reach the middle of the slice, where cells are typically patched, 5-HT needed to diffuse through a dense network of glia and other cell types. Thus, the actual concentration of 5-HT at the patched cell is likely to be lower than the concentration applied at the surface of the slice and closer to its estimated physiological concentration. The vortioxetine concentrations used here may appear to be very high when compared with the IC50 concentrations reported in the oocyte 5-HT3 receptor functional activity assays, and the 5-HT3 receptor Ki values reported above. However, there are several points that are important to consider while evaluating these data. First and foremost, the concentrations of vortioxetine, 5-HT, and mCPBG used here were chosen to be consistent with previous studies investigating other aspects of vortioxetine’s effects on hippocampal GABA and glutamate neurotransmission (Dale et al., 2014), discussed in detail below. Furthermore, neurons in JDTic 2HCl slice preparations are thought to inhabit a less favorable environment in terms of drug exposure by comparison to targets in experimental preparations such as homogenate binding and oocyte functional assays, likely due to the presence of a dense network of glia and other cellular features that could impede passive diffusion of the test compound into slices (i.e. without the usual tissue penetrating blood circulation that normally carries the compound in vivo). As a result, slice electrophysiological studies tend to use much higher concentrations than required in other assays. Moreover, this idea is reflected not only in the concentration of vortioxetine used in this experiment versus affinity for 5-HT3 receptors, but also in the concentration of 5-HT (100 µM) versus its affinity for 5-HT3 receptors (Ki = 130–320 nM; Pehrson et al., 2016a, Pehrson et al., 2016b), and ondansetron (10 µM) versus its affinity for 5-HT3 receptors (Ki = 0.36 nM; Wong et al., 1989). Perhaps most importantly, vortioxetine typically reaches total brain concentrations ranging from approximately 1 to 400 µmol/kg (approximately equivalent to 1–400 µM) in rodents at clinically-relevant dose ranges as determined by ex vivo SERT occupancy (Alan Pehrson personal communication). Thus, the 20 µM concentrations used here can be considered clinically relevant. Thus, these data support the hypothesis that vortioxetine directly suppresses 5-HT mediated excitatory drive in a subclass of GABAergic interneurons via antagonism of the 5-HT3 receptor. However, previous work has suggested that GABAergic interneurons expressing 5-HT3 receptors are a diverse set of cells, which may be differentiated by the expression of cholecystokinin, calretinin, vasoactive intestinal peptide, or neuropeptide Y (Morales and Bloom, 1997, Puig and Gulledge, 2011), and which may play a variety of roles in modulating activity in the cortex and hippocampus (McBain and Fisahn, 2001, Kubota, 2014). Thus, a demonstration that vortioxetine suppresses 5-HT3 receptor-mediated depolarizations in hippocampal GABAergic interneurons may not be sufficient on its own to suggest a downstream disinhibition of pyramidal neuron firing. But if considered in context with previous data, this hypothesis may become more convincing. We demonstrated in a previous study using slice electrophysiological recordings that identical vortioxetine concentrations suppress the frequency and amplitude of 5-HT- and mCPBG-induced inhibitory postsynaptic currents in CA1 pyramidal neurons (Dale et al., 2014) (reproduced with permission in Fig. 6). In addition, a separate research group demonstrated that acute or subchronic treatment with vortioxetine at clinically-relevant doses significantly increases in the firing rate of cortical pyramidal neurons (Riga et al., 2016, Riga et al., 2017), where 5-HT3 receptor expressing GABAergic interneurons are also present and are expected to have similar functional roles (Pehrson et al., 2016a, Pehrson et al., 2016b). Importantly, this group found that a similar increase in pyramidal neuron firing could be attained with ondansetron, either alone or in combination with the 5-HT reuptake inhibitor escitalopram. Thus, the data presented here add to a growing line of evidence that vortioxetine may indirectly disinhibit cortical and hippocampal pyramidal neuron firing.