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Astrocytes and oligodendrocytes are main players in the brain to ensure ion and neurotransmitter homeostasis, metabolic supply, and fast action potential propagation in axons. These functions are fostered by the formation of large syncytia in which mainly astrocytes and oligodendrocytes are directly coupled. Panglial networks constitute on connexin-based gap junctions in the membranes of neighboring cells that allow the passage of ions, metabolites, and currents. However, these networks are not uniform but exhibit a brain region-dependent heterogeneous connectivity influencing electrical communication and intercellular ion spread. Here, we describe different approaches to analyze gap junctional communication in acute tissue slices that can be implemented easily in most electrophysiology and imaging laboratories. These approaches include paired recordings, determination of syncytial isopotentiality, tracer coupling followed by analysis of network topography, and wide field imaging of ion sensitive dyes. These approaches are capable to reveal cellular heterogeneity causing electrical isolation of functional circuits, reduced ion-transfer between different cell types, and anisotropy of tracer coupling. With a selective or combinatory use of these methods, the results will shed light on cellular properties of glial cells and their contribution to neuronal function. [ABSTRACT FROM AUTHOR]

The voltage‐gated sodium channel (VGSC) currents in dorsal root ganglion (DRG) neurons contain mainly TTX‐sensitive (TTX‐S) and TTX‐resistant (TTX‐R) Na+ currents. Magnolol (Mag), a hydroxylated biphenyl compound isolated from the bark of Magnolia officinalis, has been well documented to exhibit analgesic effects, but its mechanism is not yet fully understood. The aim of the present study was to investigate whether the antinociceptive effects of Mag is through inhibition of Na+ currents. Na+ currents in freshly isolated mouse DRG neurons were recorded with the whole cell patch clamp technique. Results showed that Mag inhibited TTX‐S and TTX‐R Na+ currents in a concentration‐dependent manner. The IC50 values for block of TTX‐S and TTX‐R Na+ currents were 9.4 and 7.0 μmol/L, respectively. Therefore, TTX‐R Na+ current was more susceptible to Mag than TTX‐S Na+ current. For TTX‐S Na+ channel, 10 μmol/L Mag shifted the steady state inactivation curve toward more negative by 9.8 mV, without affecting the activation curve. For TTX‐R Na+ channel, 7 μmol/L Mag shifted the steady state activation and inactivation curves toward more positive and negative potentials by 6.5 and 11.7 mV, respectively. In addition, Mag significantly postponed recovery of TTX‐S and TTX‐R Na+ currents from inactivation, and produced frequency dependent blocks of both subtypes of Na+ currents. These results suggest that the inhibitory effects of Mag on Na+ channels may contribute to its analgesic effect. [ABSTRACT FROM AUTHOR]

Ivermectin (IVM) belongs to the class of macrocyclic lactones, which is used as an antiparasitic agent. At present, the researchers focus on possibility to use IVM in treatment of certain forms of cancer and viral diseases such as COVID-19. The mechanisms of IVM action are not clear. It is assumed that IVM affects chloride channels and increases cytoplasmic concentration of chloride. This study examines the effect of IVM on chloride currents induced by glycine (IGly). Experiments were carried out on isolated pyramidal neurons of the rat hippocampus with whole-cell patch clamp. A short-term (600 msec) application of IVM in a concentration of 10 μM induced a slow inward current, which persisted after washing the neurons. The low concentrations (0.1-1000 nM) of IVM did not induce any novel current, but it rapidly and reversibly reduced the peak amplitude and accelerated desensitization of IGly in a dose-dependent manner. The threshold concentrations of IVM sufficient to reduce peak amplitude of IGly and to accelerate desensitization of IGly were 100 nM and 0.1 nM, respectively. The study revealed a high sensitivity of neuronal glycine receptors to IVM. [ABSTRACT FROM AUTHOR]

Background: Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one. Methods: A patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties. Results: Confocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence. Conclusion: To our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence. [ABSTRACT FROM AUTHOR]

Background: Studying synaptic plasticity in the rat hippocampus slice is a well‐established way to analyze cellular mechanisms related to learning and memory. Different modes of recording can be used, such as extracellular field excitatory post‐synaptic potential (EPSP) and diverse patch‐clamp methods. However, most studies using these methods have examined only up to the juvenile stage of brain maturation, which is known to terminate during late adolescence/early adulthood. Moreover, several animal models of human diseases have been developed at this late stage of brain development. To study the vulnerability of adolescent rat to the cognitive impairment of alcohol, we developed a model of binge‐like exposure in which ethanol selectively abolishes low frequency stimulation (LFS)‐induced, field EPSP long‐term depression (LTD) in the rat hippocampus slice. Methods: In the present study, we sought to use whole‐cell patch‐clamp recording in the voltage‐clamp mode to further investigate the mechanisms involved in the abolition of LFS‐induced LTD in our model of binge‐like exposure in adolescent rat hippocampus slices. In addition, we investigated LFS‐induced NMDAR‐LTD and mGluR‐LTD at different ages and changed several parameters to improve the recordings. Results: Using patch‐clamp recording, LFS‐induced NMDAR‐LTD and mGluR‐LTD could be measured until 4 weeks of age, but not in older animals. Similarly, chemical mGluR‐LTD and a combined LFS‐LTD involving both N‐Methyl‐D‐Aspartate Receptor (NMDAR) and mGluR were not measured in older animals. The absence of LFS‐LTD was not due to the loss of a diffusible intracellular agent nor the voltage mode of recording or intracellular blockade of either sodium or potassium currents. In contrast to voltage‐clamp recordings, LFS‐induced LTD tested with field recordings was measured at all ages and the effects of EtOH were visible in all cases. Conclusions: We concluded that whole‐cell patch‐clamp recordings are not suitable for studying synaptic LFS‐induced LTD in rats older than 4 weeks of age and therefore cannot be used to explore electrophysiological disturbances, such as those induced by alcohol binge drinking during adolescence, which constitutes a late period of brain maturation. [ABSTRACT FROM AUTHOR]

The electrophysiological behavior of the zebrafish heart is very similar to that of the human heart. In fact, most of the genes that codify the channels and regulatory proteins required for human cardiac function have their orthologs in the zebrafish. The high fecundity, small size, and easy handling make the zebrafish embryos/larvae an interesting candidate to perform whole animal experiments within a plate, offering a reliable and low-cost alternative to replace rodents and larger mammals for the study of cardiac physiology and pathology. The employment of zebrafish embryos/larvae has widened from basic science to industry, being of particular interest for pharmacology studies, since the zebrafish embryo/larva is able to recapitulate a complete and integrated view of cardiac physiology, missed in cell culture. As in the human heart, IKr is the dominant repolarizing current and it is functional as early as 48 h post fertilization. Finally, genome editing techniques such as CRISPR/Cas9 facilitate the humanization of zebrafish embryos/larvae. These techniques allow one to replace zebrafish genes by their human orthologs, making humanized zebrafish embryos/larvae the most promising in vitro model, since it allows the recreation of human-organ-like environment, which is especially necessary in cardiac studies due to the implication of dynamic factors, electrical communication, and the paracrine signals in cardiac function.

Prenatal opioids exposure can lead to both neonatal abstinence syndrome in newborns and neurological deficits later in life. Although opioids have been well studied in general, the cellular and molecular mechanisms by which opioids affect human fetal brain development has not been well understood. In this work, we have taken advantage of a human 3D-brain cortical organoid (hCO) that facilitated enormously the investigation of early human brain development. Using imaging, immunofluorescence, multi-electrode array (MEA) and patch clamp recording techniques, we have investigated the effect of methadone, a frequently used opioid during pregnancy, on early neural development, including neuronal growth, neural network activity and synaptic transmission in hCOs. Our results demonstrated that methadone dose-dependently halted the growth of hCOs and induced organoid disintegration after a prolonged exposure. In addition, methadone dose-dependently suppressed the firing of spontaneous action potentials in hCOs and this suppression could be reversed upon methadone withdrawal in hCOs treated with lower dosages. Further investigation using patch clamp whole cell configuration revealed that, at clinically relevant concentrations, methadone decreased the frequency and amplitude of excitatory postsynaptic currents in neurons, indicating a critical role of methadone in weakening synaptic transmission in neural networks in hCOs. In addition, methadone significantly attenuated the voltage-dependent Na+ current in hCOs. We conclude that methadone interrupts neural growth and function in early brain development.

Salt sensitivity of blood pressure (BP) refers to an increase in BP following an increase in dietary salt, which is associated with increased incidence of cardiovascular disease and early death. However, decreased sodium intake also increases mortality and morbidity. Inverse salt sensitivity (ISS), defined as a paradoxical increase in BP on a low-salt diet, about 11% of the population, may be the cause of this phenomenon. The epithelial sodium channel (ENaC) is a major regulator of sodium reabsorption in the kidney. In this study, human renal tubular epithelial cells (hRTC) were cultured from the urine of phenotyped salt study participants. αENaC expression was significantly lower in ISS than salt resistant (SR) hRTC, while ENaC-like channel activity was dramatically increased by trypsin treatment in ISS cells analyzed by patch clamp. αENaC expression was also decreased under high-salt treatment and increased by aldosterone treatment in ISS cells. Moreover, the αENaC variant, rs4764586, was more prevalent in ISS. In summary, αENaC may be associated with ISS hypertension on low salt. These findings may contribute to understanding the mechanisms of ISS and low salt effect on morbidity and mortality.

Some hearing, vestibular, and vision disorders are imputable to voltage-gated Ca2+ channels of the sensory cells. These channels convey a large Ca2+ influx despite extracellular Na+ being 70-fold more concentrated than Ca2+; such high selectivity is lost in low Ca2+, and Na+ can permeate. Since the permeation properties and molecular identity of sensory Ca2+ channels are debated, in this paper, we examine the Na+ current flowing through the L- and R-type Ca2+ channels of labyrinth hair cells. Ion currents and cytosolic free Ca2+ concentrations were simultaneously monitored in whole-cell recording synchronous to fast fluorescence imaging. L-type and R-type channels were present with different densities at selected sites. In 10 nM Ca2+, the activation and deactivation time constants of the L-type Na+ current were accelerated and its maximal amplitude increased by 6-fold compared to physiological Ca2+. The deactivation of the R-type Na+ current was not accelerated, and its current amplitude increased by 2.3-fold in low Ca2+; moreover, it was partially blocked by nifedipine in a voltage- and time-dependent manner. In conclusion, L channel gating is affected by the ion species permeating the channel, and its selectivity filter binds Ca2+ more strongly than that of R channel; furthermore, external Ca2+ prevents nifedipine from perturbing the R selectivity filter.

The activity of persistent Ca²⁺ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²⁺ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²⁺ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²⁺ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²⁺ sparklets? With the use of total internal reflection fluorescence microscopy, Ca²⁺ sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²⁺ sparklets. Persistent Ca²⁺ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³⁹F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³⁹F Cav1.2c abrogated the potentiating effect of c-Src on Ca²⁺ sparklet activity. We could not detect a significant change in persistent Ca²⁺ sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets. However, our results indicate that persistent Ca²⁺ sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.

Background and purposeT16A(inh) -A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A(inh) -A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.Experimental approachSingle channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery.Key resultsIn rabbit pulmonary artery myocytes T16A(inh) -A01 (1-30 μM) inhibited single calcium (Ca(2+) )-activated chloride (Cl(-) ) channels and whole cell currents activated by 500 nM free Ca(2+) . Similar effects were observed for single Ca(2+) -activated Cl(-) channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, F(act) (10 μM), increased single channel and whole cell Ca(2+) -activated Cl(-) currents in rabbit pulmonary arteries. In isometric tension studies, T16A(inh) -A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC(50) of 1.6 μM and suppressed the methoxamine concentration-effect curve. T16A(inh) -A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 μM T16A(inh) -A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K(+) ) channel blockers. T16A(inh) -A01 (10 μM) also relaxed human visceral adipose arteries by 88 ± 3%.Conclusions and implicationsT16A(inh) -A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels.

The electrophysiological behavior of the zebrafish heart is very similar to that of the human heart. In fact, most of the genes that codify the channels and regulatory proteins required for human cardiac function have their orthologs in the zebrafish. The high fecundity, small size, and easy handling make the zebrafish embryos/larvae an interesting candidate to perform whole animal experiments within a plate, offering a reliable and low-cost alternative to replace rodents and larger mammals for the study of cardiac physiology and pathology. The employment of zebrafish embryos/larvae has widened from basic science to industry, being of particular interest for pharmacology studies, since the zebrafish embryo/larva is able to recapitulate a complete and integrated view of cardiac physiology, missed in cell culture. As in the human heart, I Kr is the dominant repolarizing current and it is functional as early as 48 h post fertilization. Finally, genome editing techniques such as CRISPR/Cas9 facilitate the humanization of zebrafish embryos/larvae. These techniques allow one to replace zebrafish genes by their human orthologs, making humanized zebrafish embryos/larvae the most promising in vitro model, since it allows the recreation of human-organ-like environment, which is especially necessary in cardiac studies due to the implication of dynamic factors, electrical communication, and the paracrine signals in cardiac function. [ABSTRACT FROM AUTHOR]

To understand the working principles of the nervous system is key to figure out its electrical activity and how this activity spreads along the neuronal network. It is therefore crucial to develop advanced techniques aimed to record in real time the electrical activity, from compartments of single neurons to populations of neurons, to understand how higher functions emerge from coordinated activity. To record from single neurons, a technique will be presented to fabricate patch pipettes able to seal on any membrane with a single glass type and whose shanks can be widened as desired. This dramatically reduces access resistance during whole-cell recording allowing fast intracellular and, if required, extracellular perfusion. To simultaneously record from many neurons, biocompatible probes will be described employing multi-electrodes made with novel technologies, based on diamond substrates. These probes also allow to synchronously record exocytosis and neuronal excitability and to stimulate neurons. Finally, to achieve even higher spatial resolution, it will be shown how voltage imaging, employing fast voltage-sensitive dyes and two-photon microscopy, is able to sample voltage oscillations in the brain spatially resolved and voltage changes in dendrites of single neurons at millisecond and micrometre resolution in awake animals. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) regulates reproduction centrally, although, the neuroanatomical basis of the process is unknown. Therefore, the putative networking of the central GLP-1 and gonadotropin-releasing hormone (GnRH) systems was addressed in male mice using whole mount immunocytochemistry and optogenetics. Enhanced antibody penetration and optical clearing procedures applied to 500–1000 µm thick basal forebrain slices allowed the simultaneous visualization of the two distinct systems in the basal forebrain. Beaded GLP-1-IR axons innervated about a quarter of GnRH neurons (23.2 ± 1.4%) forming either single or multiple contacts. GnRH dendrites received a more intense GLP-1 innervation (64.6 ± 0.03%) than perikarya (35.4 ± 0.03%). The physiological significance of the innervation was examined by optogenetic activation of channelrhodopsin-2 (ChR2)-expressing axons of preproglucagon (GCG) neurons upon the firing of GnRH neurons by patch clamp electrophysiology in acute brain slices of triple transgenic mice (Gcg-cre/ChR2/GFP-GnRH). High-frequency laser beam stimulation (20 Hz, 10 ms pulse width, 3 mW laser power) of ChR2-expressing GCG axons in the mPOA increased the firing rate of GnRH neurons (by 75 ± 17.3%, p = 0.0007). Application of the GLP-1 receptor antagonist, Exendin-3-(9-39) (1 μM), prior to the photo-stimulation, abolished the facilitatory effect. In contrast, low-frequency trains of laser pulses (0.2 Hz, 60 pulses) had no effect on the spontaneous postsynaptic currents of GnRH neurons. The findings indicate a direct wiring of GLP-1 neurons with GnRH cells which route is excitatory for the GnRH system. The pathway may relay metabolic signals to GnRH neurons and synchronize metabolism with reproduction. [ABSTRACT FROM AUTHOR]

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1 -lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (I h ). Atoh1 -lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1 -lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1 -lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Espinosa et al.)

We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i NaP ) in rat hippocampal mossy fibre boutons. Cox's method for correcting space-clamp errors was extended to the case of an isopotential compartment with attached neurites. The method was applied to voltage-ramp experiments, in which i NaP is assumed to gate instantaneously. The raw estimates of i NaP led to predicted clamp currents that were at variance with observation, hence an algorithm was devised to improve these estimates. Optionally, the method also allows an estimate of the membrane specific capacitance, although values of the axial resistivity and seal resistance must be provided. Assuming that membrane specific capacitance and axial resistivity were constant, we conclude that seal resistance continued to fall after adding TTX to the bath. This might have been attributable to a further deterioration of the seal after baseline rather than an unlikely effect of TTX. There was an increase in the membrane specific resistance in TTX. The reason for this is unknown, but it meant that i NaP could not be determined by simple subtraction. Attempts to account for i NaP with a Hodgkin-Huxley model of the transient sodium conductance met with mixed results. One thing to emerge was the importance of voltage shifts. Also, a large variability in previously reported values of transient sodium conductance in mossy fibre boutons made comparisons with our results difficult. Various other possible sources of error are discussed. Simulations suggest a role for i NaP in modulating the axonal attenuation of EPSPs. KEY POINTS: We used whole-cell patch clamp to estimate the stationary voltage dependence of persistent sodium-current density (i NaP ) in rat hippocampal mossy fibre boutons, using a KCl-based internal (pipette) solution and correcting for the liquid junction potential (2 mV). Space-clamp errors and deterioration of the patch-clamp seal during the experiment were corrected for by compartmental modelling. Attempts to account for i NaP in terms of the transient sodium conductance met with mixed results. One possibility is that the transient sodium conductance is higher in mossy fibre boutons than in the axon shaft. The analysis illustrates the need to account for various voltage shifts (Donnan potentials, liquid junction potentials and, possibly, other voltage shifts). Simulations suggest a role for i NaP in modulating the axonal attenuation of excitatory postsynaptic potentials, hence analog signalling by dentate granule cells., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Vastagh et al.)

Introduction: Automated Patch Clamp (APC) technology has become an integral element in ion channel research, drug discovery and development pipelines to overcome the use of the highly time-consuming manual patch clamp (MPC) procedures. This automated technology offers increased throughput and promises a new model in obtaining ion channel recordings, which has significant relevance to the development of novel therapies and safety profiling of candidate therapeutic compounds., Areas Covered: This article reviews the recent innovations in APC technology, including platforms, and highlights how they have facilitated usage in both industry and academia. The review also provides an overview of the ion channel research endeavors and how APC platforms have contributed to the understanding of ion channel research, pharmacological tools and therapeutics. Furthermore, the authors provide their opinion on the challenges and goals for APC technology going forward to accelerate academic research and drug discovery across a host of therapeutic areas., Expert Opinion: It is clear that APC technology has progressed drug discovery programs, specifically in the field of neuroscience and cardiovascular research. The challenge for the future is to keep pace with fundamental research and improve translation of the large datasets obtained.

AMPA-type ionotropic glutamate receptors (AMPARs) are central to various neurological processes, including memory and learning. They assemble as homo- or heterotetramers of GluA1, GluA2, GluA3, and GluA4 subunits, each consisting of an N-terminal domain (NTD), a ligand-binding domain, a transmembrane domain, and a C-terminal domain. While AMPAR gating is primarily controlled by reconfiguration in the ligand-binding domain layer, our study focuses on the NTDs, which also influence gating, yet the underlying mechanism remains enigmatic. In this investigation, we employ molecular dynamics simulations to evaluate the NTD interface strength in GluA1, GluA2, and NTD mutants GluA2-H229N and GluA1-N222H. Our findings reveal that GluA1 has a significantly weaker NTD interface than GluA2. The NTD interface of GluA2 can be weakened by a single point mutation in the NTD dimer-of-dimer interface, namely H229N, which renders GluA2 more GluA1-like. Electrophysiology recordings demonstrate that this mutation also leads to slower recovery from desensitization. Moreover, we observe that lowering the pH induces more splayed NTD states and enhances desensitization in GluA2. We hypothesized that H229 was responsible for this pH sensitivity; however, GluA2-H229N was also affected by pH, meaning that H229 is not solely responsible and that protons exert their effect across multiple domains of the AMPAR. In summary, our work unveils an allosteric connection between the NTD interface strength and AMPAR desensitization., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Human trophoblast organoids (TOs) are a three-dimensional ex vivo culture model that can be used to study various aspects of placental development, physiology and pathology. However, standard culturing of TOs does not recapitulate the cellular orientation of chorionic villi in vivo given that the multi-nucleated syncytiotrophoblast (STB) develops largely within the inner facing surfaces of these organoids (STBin). Here, we developed a method to culture TOs under conditions that recapitulate the cellular orientation of chorionic villi in vivo. We show that culturing STBin TOs in suspension with gentle agitation leads to the development of TOs containing the STB on the outer surface (STBout). Using membrane capacitance measurements, we determined that the outermost surface of STBout organoids contain large syncytia comprising >50 nuclei, whereas STBin organoids contain small syncytia (<10 nuclei) and mononuclear cells. The growth of TOs under conditions that mimic the cellular orientation of chorionic villi in vivo thus allows for the study of a variety of aspects of placental biology under physiological conditions., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Mutations of the SCN1A gene, which encodes the voltage-dependent Na + channel's α subunit, are associated with diverse epileptic syndromes ranging in severity, even intra-family, from febrile seizures to epileptic encephalopathy. The underlying cause of this variability is unknown, suggesting the involvement of additional factors. The aim of our study was to describe the properties of mutated channels and investigate genetic causes for clinical syndromes' variability in the family of five SCN1A gene p.Arg1596Cys mutation carriers. The analysis of additional genetic factors influencing SCN1A -associated phenotypes was conducted through exome sequencing (WES). To assess the impact of mutations, we used patch clamp analysis of mutated channels expressed in HEK cells and in vivo neural excitability studies (NESs). In cells expressing the mutant channel, sodium currents were reduced. NESs indicated increased excitability of peripheral motor neurons in mutation carriers. WES showed the absence of non-SCA1 pathogenic variants that could be causative of disease in the family. Variants of uncertain significance in three genes, as potential modifiers of the most severe phenotype, were identified. The p.Arg1596Cys substitution inhibits channel function, affecting steady-state inactivation kinetics. Its clinical manifestations involve not only epileptic symptoms but also increased excitability of peripheral motor fibers. The role of Nav1.1 in excitatory neurons cannot be ruled out as a significant factor of the clinical phenotype.

The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43. NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.

This paper presents a borosilicate glass micropipette device that has a high-aspect-ratio pore for patch clamp recording applications. The device was fabricated by carrying out glass reflow processes on a silicon substrate. The shape of the micropipette was determined by the width and thickness of the reflowed glass structure, which matches well with the rounded tip of a conventional glass pipette. The diameter of the micropipette pore was first defined by deep reactive ion etching (DRIE) of a silicon pillar, which was positioned at the center of the glass structure. Then, the silicon pillar was removed, and the pore diameter was additionally reduced to the submicron scale by depositing a parylene-C film. The fabricated device was integrated with microfluidic channels for patch clamp recording experiments: microfluidic PDMS layers were assembled on the top and bottom surfaces of the micropipette chip for the medium exchange and the application of negative pressure, respectively. The quality of seal between a cerebral cortex cell of a rat and the glass micropipette was characterized by the amplitude of the current produced by an applied voltage pulse. A negative pressure applied through the bottom microfluidic channel pulled down the cell and increased the seal resistance from 9.82 $\text{M}\Omega $ to 2.06 $\text{G}\Omega $. This indicates that the gigaseal was successfully formed around the micropipette pore. In addition, the action potentials of the patched cortical neuron were recorded using the fabricated device. [ABSTRACT FROM AUTHOR]

The olfactory bulb (OB) serves as a relay region for sensory information transduced by receptor neurons in the nose and ultimately routed to a variety of cortical areas. Despite the highly structured organization of the sensory inputs to the OB, even simple monomolecular odors activate large regions of the OB comprising many glomerular modules defined by afferents from different receptor neuron subtypes. OB principal cells receive their primary excitatory input from only one glomerular channel defined by inputs from one class of olfactory receptor neurons. By contrast, interneurons, such as GABAergic granule cells (GCs), integrate across multiple channels through dendodendritic inputs on their distal apical dendrites. Through their inhibitory synaptic actions, GCs appear to modulate principal cell firing to enhance olfactory discrimination, although how GCs contribute to olfactory function is not well understood. In this study, we identify a second synaptic pathway by which principal cells in the rat (both sexes) OB excite GCs by evoking potent nondepressing EPSPs (termed large-amplitude, nondendrodendritic [LANDD] EPSPs). LANDD EPSPs show little depression in response to tetanic stimulation and, therefore, can be distinguished other EPSPs that target GCs. LANDD EPSPs can be evoked by both focal stimulation near GC proximal dendrites and by activating sensory inputs in the glomerular layer in truncated GCs lacking dendrodendritic inputs. Using computational simulations, we show that LANDD EPSPs more reliably encode the duration of principal cell discharges than DD EPSPs, enabling GCs to compare contrasting versions of odor-driven activity patterns. [ABSTRACT FROM AUTHOR]

A major driver of obesity is the increasing palatability of processed foods. Although reward circuits promote the consumption of palatable food, their involvement in obesity remains unclear. The ventral pallidum (VP) is a key hub in the reward system that encodes the hedonic aspects of palatable food consumption and participates in various proposed feeding circuits. However, there is still no evidence for its involvement in developing diet-induced obesity. Here we examine, using male C57BL6/J mice and patch-clamp electrophysiology, how chronic high-fat high-sugar (HFHS) diet changes the physiology of the VP and whether mice that gain the most weight differ in their VP physiology from others. We found that 10-12 weeks of HFHS diet hyperpolarized and decreased the firing rate of VP neurons without a major change in synaptic inhibitory input. Within the HFHS group, the top 33% weight gainers (WGs) had a more hyperpolarized VP with longer latency to fire action potentials on depolarization compared with bottom 33% of weight gainers (i.e., non-weight gainers). WGs also showed synaptic potentiation of inhibitory inputs both at the millisecond and minute ranges. Moreover, we found that the tendency to potentiate the inhibitory inputs to the VP might exist in overeating mice even before exposure to HFHS, thus making it a potential property of being an overeater. These data point to the VP as a critical player in obesity and suggest that hyperpolarized membrane potential of, and potentiated inhibitory inputs to, VP neurons may play a significant role in promoting the overeating of palatable food. [ABSTRACT FROM AUTHOR]

The identification of a pathogenic SCN5A variant confers an increased risk of conduction defects and ventricular arrhythmias (VA) in Brugada syndrome (BrS). However, specific aspects of sodium channel function that influence clinical phenotype have not been defined. A systematic literature search identified SCN5A variants associated with BrS. Sodium current (INa) functional parameters (peak current, decay, steady‐state activation and inactivation, and recovery from inactivation) and clinical features (conduction abnormalities [CA], spontaneous VA or family history of sudden cardiac death [SCD], and spontaneous BrS electrocardiogram [ECG]) were extracted. A total of 561 SCN5A variants associated with BrS were identified, for which data on channel function and clinical phenotype were available in 142. In the primary analysis, no relationship was found between any aspect of channel function and CA, VA/SCD, or spontaneous BrS ECG pattern. Sensitivity analyses including only variants graded pathogenic or likely pathogenic suggested that reduction in peak current and positive shift in steady‐state activation were weakly associated with CA and VA/SCD, although sensitivity and specificity remained low. The relationship between in vitro assessment of channel function and BrS clinical phenotype is weak. The assessment of channel function does not enhance risk stratification. Caution is needed when extrapolating functional testing to the likelihood of variant pathogenicity. [ABSTRACT FROM AUTHOR]

The retina and the olfactory bulb are the gateways to the visual and olfactory systems, respectively, similarly using neural networks to initiate sensory signal processing. Sensory receptors receive signals that are transmitted to neural networks before projecting to primary cortices. These networks filter sensory signals based on their unique features and adjust their sensitivities by gain control systems. Interestingly, dopamine modulates sensory signal transduction in both systems. In the retina, dopamine adjusts the retinal network for daylight conditions (“light adaptation”). In the olfactory system, dopamine mediates lateral inhibition between the glomeruli, resulting in odorant signal decorrelation and discrimination. While dopamine is essential for signal discrimination in the olfactory system, it is not understood whether dopamine has similar roles in visual signal processing in the retina. To elucidate dopaminergic effects on visual processing, we conducted patch-clamp recording from second-order retinal bipolar cells, which exhibit multiple types that can convey different temporal features of light. We recorded excitatory postsynaptic potentials (EPSPs) evoked by various frequencies of sinusoidal light in the absence and presence of a dopamine receptor 1 (D1R) agonist or antagonist. Application of a D1R agonist, SKF-38393, shifted the peak temporal responses toward higher frequencies in a subset of bipolar cells. In contrast, a D1R antagonist, SCH-23390, reversed the effects of SKF on these types of bipolar cells. To examine the mechanism of dopaminergic modulation, we recorded voltage-gated currents, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and low-voltage activated (LVA) Ca2+ channels. SKF modulated HCN and LVA currents, suggesting that these channels are the target of D1R signaling to modulate visual signaling in these bipolar cells. Taken together, we found that dopamine modulates the temporal tuning of a subset of retinal bipolar cells. Consequently, we determined that dopamine plays a role in visual signal processing, which is similar to its role in signal decorrelation in the olfactory bulb.

ABSTRACT Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

The superficial dorsal horn (SDH, LI-II) of the spinal cord receives and processes multimodal sensory information from skin, muscle, joints, and viscera then relay it to the brain. Neurons within the SDH fall into two broad categories, projection neurons and interneurons. The later can be further subdivided into excitatory and inhibitory types. Traditionally, interneurons within the SDH have been divided into overlapping groups according to their neurochemical, morphological and electrophysiological properties. Recent clustering analyses, based on molecular transcript profiles of cells and nuclei, have predicted many more functional groups of interneurons than expected using traditional approaches. In this study, we used electrophysiological and morphological data obtained from genetically-identified excitatory (vGLUT2) and inhibitory (vGAT) interneurons in transgenic mice to cluster cells into groups sharing common characteristics and subsequently determined how many clusters can be assigned by combinations of these properties. Consistent with previous reports, we show differences exist between excitatory and inhibitory interneurons in terms of their excitability, nature of the ongoing excitatory drive, action potential (AP) properties, sub-threshold current kinetics, and morphology. The resulting clusters based on statistical and unbiased assortment of these data fell well short of the numbers of molecularly predicted clusters. There was no clear characteristic that in isolation defined a population, rather multiple variables were needed to predict cluster membership. Importantly though, our analysis highlighted the appropriateness of using transgenic lines as tools to functionally subdivide both excitatory and inhibitory interneuron populations.

Type III and IV Bartter syndromes (BS) are rare kidney tubulopathies caused by loss-of-function mutations in the CLCNKB and BSND genes coding respectively for the ClC-Kb chloride channels and accessory subunit barttin. ClC-K channels are expressed in the Henle’s loop, distal convoluted tubule, and cortical collecting ducts of the kidney and contribute to chloride absorption and urine concentration. In our Italian cohort, we identified two new mutations in CLCNKB, G167V and G289R, in children affected by BS and previously reported genetic variants, A242E, a chimeric gene and the deletion of the whole CLCNKB. All the patients had hypokalemia and metabolic alkalosis, increased serum renin and aldosterone levels and were treated with a symptomatic therapy. In order to define the molecular mechanisms responsible for BS, we co-expressed ClC-Kb wild type and channels with point mutations with barttin in HEK 293 cells and characterized chloride currents through the patch-clamp technique. In addition, we attempted to revert the functional defect caused by BS mutations through barttin overexpression. G167V and A242E channels showed a drastic current reduction compared to wild type, likely suggesting compromised expression of mutant channels at the plasma membrane. Conversely, G289R channel was similar to wild type raising the doubt that an additional mutation in another gene or other mechanisms could account for the clinical phenotype. Interestingly, increasing ClC-K/barttin ratio augmented G167V and A242E mutants’ chloride current amplitudes towards wild type levels. These results confirm a genotype-phenotype correlation in BS and represent a preliminary proof of concept that molecules functioning as molecular chaperones can restore channel function in expression-defective ClC-Kb mutants.

Behavioral studies have shown spatial working memory impairment with aging in several animal species, including humans. Persistent activity of layer 3 pyramidal dorsolateral prefrontal cortex (dlPFC) neurons during delay periods of working memory tasks is important for encoding memory of the stimulus. In vitro studies have shown that these neurons undergo significant age-related structural and functional changes, but the extent to which these changes affect neural mechanisms underlying spatial working memory is not understood fully. Here, we confirm previous studies showing impairment on the Delayed Recognition Span Task in the spatial condition (DRSTsp), and increased in vitro action potential firing rates (hyperexcitability), across the adult life span of the rhesus monkey. We use a bump attractor model to predict how empirically observed changes in the aging dlPFC affect performance on the Delayed Response Task (DRT), and introduce a model of memory retention in the DRSTsp. Persistent activity—and, in turn, cognitive performance—in both models was affected much more by hyperexcitability of pyramidal neurons than by a loss of synapses. Our DRT simulations predict that additional changes to the network, such as increased firing of inhibitory interneurons, are needed to account for lower firing rates during the DRT with aging reported in vivo. Synaptic facilitation was an essential feature of the DRSTsp model, but it did not compensate fully for the effects of the other age-related changes on DRT performance. Modeling pyramidal neuron hyperexcitability and synapse loss simultaneously led to a partial recovery of function in both tasks, with the simulated level of DRSTsp impairment similar to that observed in aging monkeys. This modeling work integrates empirical data across multiple scales, from synapse counts to cognitive testing, to further our understanding of aging in non-human primates.

Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.

Anna Binda,1 Alice Panariti,1 Andrea Barbuti,2 Carmen Murano,1 Roberta Dal Magro,1 Massimo Masserini,1,3,4 Francesca Re,1,3,4 Ilaria Rivolta,1,3,4 1School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy; 2Department of Biosciences, The PaceLab and Interuniversity Center of Molecular Medicine and Applied Biophysics (CIMMBA), University of Milan, Milano, Italy; 3Milan Center for Neuroscience (NeuroMI), University of Milano-Bicocca, Monza, Italy; 4Nanomedicine Center NANOMIB, University of Milano-Bicocca, Milano, Italy Purpose: Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. Materials and methods: We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood–brain barrier and to be effective on mouse models of Alzheimer’s disease (AD), both in the absence and in the presence of β-amyloid peptide oligomers. Results: Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with β-amyloid peptide oligomers suggest a retention of liposome efficacy. Conclusion: These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability. Keywords: neurodegenerative disorders, nanomedicine, action potential, electrophysiology, patch clamp, β-amyloid peptide

AIM: To evaluate the intrinsic excitability of retinal ganglion cells (RGCs) in degenerated retinas. METHODS: The intrinsic excitability of various morphologically defined RGC types using a combination of patch-clamp recording and the Lucifer yellow tracer in retinal whole-mount preparations harvested from Royal College of Surgeons (RCS) rats, a common retinitis pigmentosa (RP) model, in a relatively late stage of retinal degeneration (P90) were investigated. Several parameters of RGC morphologies and action potentials (APs) were measured and compared to those of non-dystrophic control rats, including dendritic stratification, dendritic field diameter, peak amplitude, half width, resting membrane potential, AP threshold, depolarization to threshold, and firing rates. RESULTS: Compared with non-dystrophic control RGCs, more depolarizations were required to reach the AP threshold in RCS RGCs with low spontaneous spike rates and in RCS OFF cells (especially A2o cells), and RCS RGCs maintained their dendritic morphologies, resting membrane potentials and capabilities to generate APs. CONCLUSION: RGCs are relatively well preserved morphologically and functionally, and some cells are more susceptible to decreased excitability during retinal degeneration. These findings provide valuable considerations for optimizing RP therapeutic strategies.

Background/Aims: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. Methods: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. Results: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. Conclusions: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (Mecp2) gene. GABAergic dysfunction has been implicated contributing to the respiratory dysfunction, one major clinical feature of RTT. The nucleus tractus solitarius (NTS) is the first central site integrating respiratory sensory information that can change the nature of the reflex output. We hypothesized that deficiency in Mecp2 gene reduces GABAergic neurotransmission in the NTS. Using whole-cell patch-clamp recordings in NTS slices, we measured spontaneous inhibitory postsynaptic currents (sIPSCs), miniature IPSCs (mIPSCs), NTS-evoked IPSCs (eIPSCs), and GABAA receptor (GABAA-R) agonist-induced responses. Compared to those from wild-type mice, NTS neurons from Mecp2-null mice had significantly (p

• Prenatal nicotine alters development of acidosis response in medullary 5-HT neurons. • Prenatal nicotine blunts the acidosis response of cultured medullary 5-HT neurons. • Prenatal nicotine blunts the effects of acute nicotine on medullary 5-HT neurons. • Impaired function of 5-HT neurons through prenatal nicotine may increase SIDS risk. Perinatal exposure to nicotine produces ventilatory and chemoreflex deficits in neonatal mammals. Medullary 5-HT neurons are putative central chemoreceptors that innervate respiratory nuclei and promote ventilation, receive cholinergic input and express nicotinic acetylcholine receptors (nAChRs). Perforated patch clamp recordings were made from cultured 5-HT neurons dissociated from the medullary raphé of 0–3 day old mice expressing enhanced yellow fluorescent protein driven by the enhancer region for PET1 (ePet-EYFP). The effect of exposure to low (6 mg kg−1day−1) or high (60 mg kg−1day−1) doses of nicotine in utero (prenatal), in culture (postnatal), or both and the effect of acute nicotine exposure (10 μM), were examined on baseline firing rate (FR at 5% CO 2 , pH = 7.4) and the change in FR with acidosis (9% CO 2 , pH 7.2) in young (12–21 days in vitro , DIV) and older (≥22 DIV) acidosis stimulated 5-HT neurons. Nicotine exposed neurons exhibited ∼67% of the response to acidosis recorded in neurons given vehicle (p = 0.005), with older neurons exposed to high dose prenatal and postnatal nicotine, exhibiting only 28% of that recorded in the vehicle neurons (p < 0.01). In neurons exposed to low or high dose prenatal and postnatal nicotine , acute nicotine exposure led to a smaller increase in FR (∼+51% vs +168%, p = 0.026) and response to acidosis (+6% vs +67%, p = 0.014) compared to vehicle. These data show that exposure to nicotine during development reduces chemosensitivity of 5-HT neurons as they mature, an effect that may be related to the abnormal chemoreflexes reported in rodents exposed to nicotine in utero , and may cause a greater risk for sudden infant death syndrome (SIDS). [ABSTRACT FROM AUTHOR]

Conversion of mesenchymal stem cells (MSC) into neuron-like cells (NLC) is a feasible cell therapy strategy for replacing lost neurons in neuronal disorders. In this study, adipose-derived MSC (ADMSC) were converted into neural stem cells (NSC) via neurosphere. The resulting NSC were then differentiated into NLC by transduction with microRNA-218, using a lentiviral vector. ADMSC, NSC, and NLC were first characterized by flow cytometry, RT-PCR, and immunocytochemistry. The functionality of the NLC was evaluated by qRT-PCR and patch clamp recording. Immunophenotyping of ADMSC showed their immunoreactivity to MSC markers CD90, CD73, CD105, and CD49d, but not to CD31 and CD45. RT-PCR results demonstrated the expression of nestin, neurogenin, neurod1, neurofilament light, and GAP43 genes in NSC while NLC expressed synaptophysin, neurofilament heavy, and GAP43. In addition, NSC morphology changed into multipolar with long processes after transduction with miR-218. Moreover, using qRT-PCR, the expression levels of miR-218 and functionality genes CACNA1C, SNAP25, KCNH1, KCNMA1, and SCN9A were significantly increased in NLC, compared with NSC, and ADMSC at 3 weeks and 5 months post-transduction. Furthermore, the generated NLC expressed significantly higher protein levels of neurofilament heavy polypeptide (NFh) and enolase 2 (Eno2) neuronal markers, compared with ADMSC and NSC. Finally, action potentials were successfully recorded by the generated NLC, using patch clamp. In summary, ADMSC-derived NSC differentiated into functional NLC by transduction with miR-218. The generated NLC expressed functional SNAP25, CACNA1C, KCNH1, KCNMA1, and SCN9A and produced an action potential, which provides useful insights into the generation of functional neuronal cells. [ABSTRACT FROM AUTHOR]

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BACKGROUND: Opioids can regulate the changes of membrane potential and Ca2+ current in cardiomyocytes, but whether diacetylmorphine can induce the changes of cardiac rhythm, cell action potential and Ca2+ current has not been reported. OBJECTIVE: To explore the effect of diacetylmorphine on action potential and calcium current of isolated cardiomyocytes from neonatal Sprague-Dawley rats. METHODS: Five concentrations of diacetylmorphine (0, 10-2, 10-3, 10-4, 10-5 mol/L) and 20 mol/L verapamil were used to treat the cardiomyocytes of neonatal Sprague-Dawley rats cultured in vitro. The cells were divided into control group, diacetylmorphine group, diacetylmorphine+verapamil group. The latter two groups were treated with diacetylmorphine and diacetylmorphine+verapamil (20 μmol/L), respectively, while the control group was treated with the same amount of PBS. The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University on May 21, 2018 with approval No. IACUC201805-K1. RESULTS AND CONCLUSION: At 24 hours of culture with different concentrations of diacetylmorphine, the number of cardiomyocytes with abnormal morphology increased significantly in a dose-dependent manner. When the concentration of diacetylmorphine increased, the number of survived cells decreased, with a reduction in the size of cytoplasm and number of pseudopods, the cell membrane was shrunk and the nuclear structure was blurred. Compared with the control group, when diacetylmorphine was added to intervene with the cardiomyocytes, there was a significant difference in the spontaneous beating frequency and rhythm of cardiomyocytes. The negative value of resting membrane potential decreased, while the time course of action potential increased significantly, and the amplitude of action potential decreased significantly. Compared with the control group, the number of cells with changes in the membrane potential significantly increased in the diacetylmorphine group. The addition of verapamil reduced the number of cells with changes in the membrane potential. Compared with the control group, the number of cells with variation of membrane potential was increased to some extents. These findings suggest that diacetylmorphine can induce cardiomyocyte morphological abnormality, increase the spontaneous beating frequency and rhythm of cardiomyocytes, and change the membrane potential and action potential of cardiomyocytes. Verapamil acts as a calcium channel blocker that can improve the rhythm abnormality of cardiomyocytes induced by diacetylmorphine. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of miR-34c-5p on the main voltage-dependent ion channels in skeletal muscle cells. This study focused on the effects of miR-34c-5p on sodium, potassium, and calcium currents in C2C12 myoblasts. The miR-34c-5p overexpression group, knockdown group, and control group were differentiated for 7 days, fused into myotubes, and used for the whole-cell patch clamp recording. Compared with the control group, the whole-cell sodium current density of the other two groups had no significant changes. In the knockdown group, the delayed rectifier potassium current density was increased (statistically significant), and the whole-cell calcium channel current density did not change. In the overexpression group, the change of rectifier potassium current density was not obvious, while the peak calcium channel current density increased (− 9.23 ± 0.95 pA/pF, n = 6 cells for the overexpression group vs. − 6.48 ± 0.64 pA/pF, n = 7 cells for the control; p < 0.05). Changes in the expression of miR-34c-5p can affect the electrophysiological characteristics of calcium and potassium voltage-gated channels in C2C12 myotubes. Overexpression of miR-34c-5p increased whole-cell L-type calcium channel current (ICa,L), while miR-34c-5p knockdown increased whole-cell delayed rectifier potassium current (IKd). [ABSTRACT FROM AUTHOR]

Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-l-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis. [ABSTRACT FROM AUTHOR]

The retina and the olfactory bulb are the gateways to the visual and olfactory systems, respectively, similarly using neural networks to initiate sensory signal processing. Sensory receptors receive signals that are transmitted to neural networks before projecting to primary cortices. These networks filter sensory signals based on their unique features and adjust their sensitivities by gain control systems. Interestingly, dopamine modulates sensory signal transduction in both systems. In the retina, dopamine adjusts the retinal network for daylight conditions ("light adaptation"). In the olfactory system, dopamine mediates lateral inhibition between the glomeruli, resulting in odorant signal decorrelation and discrimination. While dopamine is essential for signal discrimination in the olfactory system, it is not understood whether dopamine has similar roles in visual signal processing in the retina. To elucidate dopaminergic effects on visual processing, we conducted patch-clamp recording from second-order retinal bipolar cells, which exhibit multiple types that can convey different temporal features of light. We recorded excitatory postsynaptic potentials (EPSPs) evoked by various frequencies of sinusoidal light in the absence and presence of a dopamine receptor 1 (D1R) agonist or antagonist. Application of a D1R agonist, SKF-38393, shifted the peak temporal responses toward higher frequencies in a subset of bipolar cells. In contrast, a D1R antagonist, SCH-23390, reversed the effects of SKF on these types of bipolar cells. To examine the mechanism of dopaminergic modulation, we recorded voltage-gated currents, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and low-voltage activated (LVA) Ca2+ channels. SKF modulated HCN and LVA currents, suggesting that these channels are the target of D1R signaling to modulate visual signaling in these bipolar cells. Taken together, we found that dopamine modulates the temporal tuning of a subset of retinal bipolar cells. Consequently, we determined that dopamine plays a role in visual signal processing, which is similar to its role in signal decorrelation in the olfactory bulb. [ABSTRACT FROM AUTHOR]

Inhibitory interneurons in the adult spinal dorsal horn (DH) can be neurochemically classified into subpopulations that regulate distinct somatosensory modalities. Although inhibitory networks in the rodent DH undergo dramatic remodeling over the first weeks of life, little is known about the maturation of identified classes of GABAergic interneurons, or whether their role in somatosensation shifts during development. We investigated age-dependent changes in the connectivity and function of prodynorphin (DYN)-lineage neurons in the mouse DH that suppress mechanosensation and itch during adulthood. In vitro patch clamp recordings revealed a developmental increase in primary afferent drive to DYN interneurons and a transition from exclusive C-fiber monosynaptic input to mixed A- and C-fiber innervation. While most adult DYN interneurons exhibited tonic firing as expected from their inhibitory phenotype, neonatal and adolescent DYN cells were predominantly classified as phasic or single-spiking. Importantly, we also found that the majority of inhibitory presynaptic terminals contacting lamina I spinoparabrachial projection neurons (PNs) originate from DYN neurons. Furthermore, inhibitory synaptic input from DYN interneurons onto PNs was weaker during the neonatal period, likely reflecting a lower number of GABAergic terminals and a reduced probability of GABA release compared to adults. Finally, spinal DYN interneurons attenuated mechanical sensitivity throughout development, but this population dampened acute non-histaminergic itch only during adulthood. Collectively, these findings suggest that the spinal 'gates' controlling sensory transmission to the brain may emerge in a modality-selective manner during early life due to the postnatal tuning of inhibitory synaptic circuits within the DH. [ABSTRACT FROM AUTHOR]

Neural circuits require balanced synaptic excitation and inhibition to ensure accurate neural computation. Our knowledge about the development and maturation of inhibitory synaptic inputs is less well developed than that concerning excitation. Here we describe the maturation of an inhibitory circuit within the mammalian auditory brainstem where counterintuitively, inhibition drives action potential firing of principal neurons. With the use of combined anatomical tracing and electrophysiological recordings from mice, neurons of the superior paraolivary nucleus (SPN) are shown to receive converging glycinergic input from at least four neurons of the medial nucleus of the trapezoid body (MNTB). These four axons formed 30.71 ± 2.72 (means ± SE) synaptic boutons onto each SPN neuronal soma, generating a total inhibitory conductance of 80 nS. Such strong inhibition drives the underlying postinhibitory rebound firing mechanism, which is a hallmark of SPN physiology. In contrast to inhibitory projections to the medial and lateral superior olives, the inhibitory projection to the SPN does not exhibit experience-dependent synaptic refinement following the onset of hearing. These findings emphasize that the development and function of neural circuits cannot be inferred from one synaptic target to another, even if both originate from the same neuron. NEW & NOTEWORTHY Neuronal activity regulates development and maturation of neural circuits. This activity can include spontaneous burst firing or firing elicited by sensory input during early development. For example, auditory brainstem circuits involved in sound localization require acoustically evoked activity to form properly. Here we show, that an inhibitory circuit, involved in processing sound offsets, gaps, and rhythmically modulated vocal communication signals, matures before the onset of acoustically evoked activity. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluNl (G638; 1642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B Ml helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the Ml and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluNl M3 helix residues G638 and 1642. This stabilizes the open-state position of the GluNl M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented. [ABSTRACT FROM AUTHOR]

Partial or complete loss-of-function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada syndrome-associated variants, selected on the basis of their frequency in the general population and in individuals with Brugada syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%–50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n = 2), likely pathogenic (n = 10), or VUSs (n = 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A. [ABSTRACT FROM AUTHOR]

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved. [ABSTRACT FROM AUTHOR]