Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca²⁺ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca²⁺ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca²⁺-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca²⁺-dependent proarrhythmic models, freely moving Trpm4^−/− mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca²⁺ overload-induced background current, thereby regulating cell excitability in Ca²⁺ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4^−/− mice, 10 µM meclofenamate inhibited the Ca²⁺ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca²⁺-dependent triggered arrhythmias. Proposed mechanism of the specific contribution of TRPM4 to the cellular mechanism of Ca²⁺-dependent triggered arrhythmias associated with, for example, acute ischaemia or catecholaminergic polymorphic ventricular tachycardia (CPVT). Typically for these conditions, aberrant Ca²⁺ release events induce a transient inward current (I_ti) on top of a background current (I_background) in ventricular myocytes, which underlies delayed afterdepolarizations that trigger ectopic action potentials when the threshold potential is reached. The TRPM4 specifically contributes to the Ca²⁺ overload-induced background current that augments cellular excitability, and inhibition of TRPM4 with meclofenamate efficiently suppresses CPVT-associated arrhythmias in living mice. AP, action potential; CPVT, catecholaminergic polymorphic ventricular tachycardia; DAD, delayed afterdepolarization; V_m, membrane potential; V_threshold, threshold potential.

Somatostatin is an important mood and pain-regulating neuropeptide, which exerts analgesic, anti-inflammatory, and antidepressant effects via its Gi protein-coupled receptor subtype 4 (SST₄) without endocrine actions. SST₄ is suggested to be a unique novel drug target for chronic neuropathic pain, and depression, as a common comorbidity. However, its neuronal expression and cellular mechanism are poorly understood. Therefore, our goals were (i) to elucidate the expression pattern of Sstr4/SSTR4 mRNA, (ii) to characterize neurochemically, and (iii) electrophysiologically the Sstr4/SSTR4-expressing neuronal populations in the mouse and human brains. Here, we describe SST₄ expression pattern in the nuclei of the mouse nociceptive and anti-nociceptive pathways as well as in human brain regions, and provide neurochemical and electrophysiological characterization of the SST₄-expressing neurons. Intense or moderate SST₄ expression was demonstrated predominantly in glutamatergic neurons in the major components of the pain matrix mostly also involved in mood regulation. The SST₄ agonist J-2156 significantly decreased the firing rate of layer V pyramidal neurons by augmenting the depolarization-activated, non-inactivating K⁺ current (M-current) leading to remarkable inhibition. These are the first translational results explaining the mechanisms of action of SST₄ agonists as novel analgesic and antidepressant candidates.

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons and regulate nociceptor and inflammatory functions. Resolvins are endogenous lipid mediators. Resolvin D1 (RvD1) is described as a selective inhibitor of TRPA1-related postoperative and inflammatory pain in mice acting on the G protein-coupled receptor DRV1/GPR32. Resolvin D2 (RvD2) is a very potent TRPV1 and TRPA1 inhibitor in DRG neurons, and decreases inflammatory pain in mice acting on the GPR18 receptor, via TRPV1/TRPA1-independent mechanisms. We provided evidence that resolvins inhibited neuropeptide release from the stimulated sensory nerve terminals by TRPV1 and TRPA1 activators capsaicin (CAPS) and allyl-isothiocyanate (AITC), respectively. We showed that RvD1 and RvD2 in nanomolar concentrations significantly decreased TRPV1 and TRPA1 activation on sensory neurons by fluorescent calcium imaging and inhibited the CAPS- and AITC-evoked ⁴⁵Ca-uptake on TRPV1- and TRPA1-expressing CHO cells. Since CHO cells are unlikely to express resolvin receptors, resolvins are suggested to inhibit channel opening through surrounding lipid raft disruption. Here, we proved the ability of resolvins to alter the membrane polarity related to cholesterol composition by fluorescence spectroscopy. It is concluded that targeting lipid raft integrity can open novel peripheral analgesic opportunities by decreasing the activation of nociceptors.

GABA released from heterogeneous types of interneurons acts in a complex spatio-temporal manner on postsynaptic targets in the networks. In addition to GABA, a large fraction of GABAergic cells also express neuromodulator peptides. Somatostatin (SOM) containing interneurons, in particular, have been recognized as key players in several brain circuits, however, the action of SOM and its downstream network effects remain largely unknown. Here, we used optogenetics, electrophysiologic, anatomical and behavioral experiments to reveal that the dendrite-targeting, SOM⁺ GABAergic interneurons demonstrate a unique layer-specific action in the medial entorhinal cortex (MEC) both in terms of GABAergic and SOM-related properties. We show that GABAergic and somatostatinergic neurotransmission originating from SOM⁺ local interneurons preferentially inhibit layer_III-V pyramidal cells, known to be involved in memory formation. We propose that this dendritic GABA–SOM dual inhibitory network motif within the MEC serves to selectively modulate working-memory formation without affecting the retrieval of already learned spatial navigation tasks.

Introduction:The centrally projecting Edinger-Westphal nucleus (EWcp) contributes to the control of alcohol consumption by its urocortin 1 (UCN1) and cocaine- and amphetamine-regulated transcript (CART) co-expressing peptidergic neurons. Our group recently showed that the urocortinergic centrally projecting EWcp is the primary seat of central nervous system transient receptor potential ankyrin 1 (TRPA1) cation channel mRNA expression. Here, we hypothesized that alcohol and its metabolites, that pass through the blood-brain barrier, may influence the function of urocortinergic cells in centrally projecting EWcp by activating TRPA1 ion channels. We aimed to examine the functional activity of TRPA1 in centrally projecting EWcp and its possible role in a mouse model of acute alcohol exposure.Methods:Electrophysiological measurements were performed on acute brain slices of C57BL/6J male mice containing the centrally projecting EWcp to prove the functional activity of TRPA1 using a selective, potent, covalent agonist JT010. Male TRPA1 knockout (KO) and wildtype (WT) mice were compared with each other in the morphological studies upon acute alcohol treatment. In both genotypes, half of the animals was treated intraperitoneally with 1 g/kg 6% ethanol vs. physiological saline-injected controls. Transcardial perfusion was performed 2 h after the treatment. In the centrally projecting EWcp area, FOS immunohistochemistry was performed to assess neuronal activation. TRPA1, CART, and urocortin 1 mRNA expression as well as urocortin 1 and CART peptide content was semi-quantified by RNAscopein situhybridization combined with immunofluorescence.Results:JT010 activated TRPA1 channels of the urocortinergic cells in acute brain slices. Alcohol treatment resulted in a significant FOS activation in both genotypes. Alcohol decreased theTrpa1mRNA expression in WT mice. The assessment of urocortin 1 peptide immunoreactivity revealed lower basal urocortin 1 in KO mice compared to WTs. The urocortin 1 peptide content was affected genotype-dependently by alcohol: the peptide content decreased in WTs while it increased in KO mice. Alcohol exposure influenced neither CART and urocortin 1 mRNA expression nor the centrally projecting EWcp/CART peptide content.Conclusion:We proved the presence of functional TRPA1 receptors on urocortin 1 neurons of the centrally projecting EWcp. DecreasedTrpa1mRNA expression upon acute alcohol treatment, associated with reduced neuronal urocortin 1 peptide content suggesting that this cation channel may contribute to the regulation of the urocortin 1 release.

Background: Mossy cells comprise a large fraction of excitatory neurons in the hippocampal dentate gyrus, and their loss is one of the major hallmarks of temporal lobe epilepsy (TLE). The vulnerability of mossy cells in TLE is well known in animal models as well as in patients; however, the mechanisms leading to cellular death is unclear. Results: Transient receptor potential melastatin 4 (TRPM4) is a Ca²⁺-activated non-selective cation channel regulating diverse physiological functions of excitable cells. Here, we identified that TRPM4 is present in hilar mossy cells and regulates their intrinsic electrophysiological properties including spontaneous activity and action potential dynamics. Furthermore, we showed that TRPM4 contributes to mossy cells death following status epilepticus and therefore modulates seizure susceptibility and epilepsy-related memory deficits. Conclusions: Our results provide evidence for the role of TRPM4 in MC excitability both in physiological and pathological conditions.