Analysis of mitochondrial translocases TOM and TIM by the patch-clamping technique.

Mitochondrial protein import and sorting relies on sophisticated molecular machineries or translocases, of which channels are integral. Channels are built upon membrane proteins whose functions are driven by conformational changes. This implies that structural and functional information need to be integrated to gain a deep understanding of their dynamic behavior. Patch-clamp approaches are well suited for this purpose. This chapter provides a detailed description and practical guidance for applying the patch-clamp methodology to the electrophysiological characterization of mitochondrial protein import. Implementing the technique to intact mitochondria, mitoplasts, and reconstituted proteoliposomes, combined with genetically modified yeast strains, expands the scope of these studies. Focused on the TOM, TIM23, and TIM22 translocases, an analysis of the patch-clamp contribution to the field is outlined.

Adrenomedullin-mediated depressor response with visceral afferent-specific membrane depolarization in isolated nodose ganglion neurons from adult female rat.

Adrenomedullin (ADM) is an endogenous and vasoactive neuropeptide that possesses potent central/peripheral regulations on blood pressure (BP) and sex-related vasodilation under physiological conditions. However, the role of ADM on baroreflex afferent function is largely unknown. Here, BP was monitored in adult female rats while ADM was microinjected into the nodose ganglion (NG); Fluorescent intensity against ADM was analyzed in the tissue level and membrane responses elicited by ADM were tested in identified NG neurons isolated from adult female rats with gap-free protocol under current-clamp mode with or without ADM antagonist. The results showed that BP was reduced by ADM (30-300 nM) concentration-dependently; myelinated (HCN1-positive) neurons showed significantly higher fluorescent intensity against ADM antibody vs. unmyelinated (HCN1-negative) neurons. Interestingly, patch-clamp data indicated that membrane potential was not changed in 50 % (6/12) of identified A-types, only 4/12 was hyperpolarized by 30 nM ADM, while 100 nM ADM induced brief hyperpolarization followed by depolarization in 2/12 of recordings; Robustly, ADM depolarized 100 % tested myelinated Ah-type neurons with dramatic and concentration-dependent repetitive discharges; While, a majority (8/9) of unmyelinated C-types were depolarized and few with repetitive dischargers. By application of ADM (22-52), the depolarization elicited by ADM 100 nM was partially or completely abolished in Ah-types or C-types, respectively. These datasets demonstrated for the first time that baroreflex afferents especially female-distributed subpopulation of Ah-types would be a key player in ADM-mediated depressor response unveiling the dominate role of peripheral ADM in neurocontrol of hypotension via baroreflex afferent function and gender-dependent vasodilation promoted by female sex steroid.

Progress and potential of brain organoids in epilepsy research.

Epilepsies are disorders of the brain characterised by an imbalance in electrical activity, linked to a disruption in the excitation and inhibition of neurons. Progress in the epilepsy research field has been hindered by the lack of an appropriate model, with traditionally used 2D primary cell culture assays and animal models having a number of limitations which inhibit their ability to recapitulate the developing brain and the mechanisms behind epileptogenesis. As a result, the mechanisms behind the pathogenesis of epilepsy are largely unknown. Brain organoids are 3D aggregates of neural tissue formed in vitro and have been shown to recapitulate the gene expression patterns of the brain during development, and can successfully model a range of epilepsies and drug responses. They thus present themselves as a novel tool to advance studies into epileptogenesis. In this review, we discuss the formation of brain organoids, their recent application in studying genetic epilepsies, hyperexcitability dynamics and oxygen glucose deprivation as a hyperexcitability agent, their use as an epilepsy drug testing and development platform, as well as the limitations of their use in epilepsy research and how these can be mitigated.

Evaluating the proarrhythmic risk of delayed-action compounds in serum free cell culture conditions; serum-starvation accelerates/amplifies the effect of probucol on the KCNQ1 + KCNE1 channel.

In vitro testing procedures for evaluating acute effects of compound on ion channels, utilizing heterologous expression systems (HES), are well-established, while slowly manifesting delayed effects remain challenging to detect. For this, immortalized HES are exposed to the compounds for a longer time, in general 24 h. As these cells proliferate every 12-20 h, we evaluated if the proliferation status, and by extension cell metabolism, influences the delayed compound response. The intervention of halting cell proliferation by excluding serum from the culturing medium was evaluated on CHO cells, stably expressing the KCNQ1 + KCNE1 channel complex that mediates the slow delayed rectifier potassium current (Iks). No abnormal changes in KCNQ1 + KCNE1 current were observed upon serum-starvation, except for a negative shift in the voltage dependence of channel activation (GV-curve) after 72 h. The delayed effect of probucol, a compound reported to interfere with Iks expression, was evaluated after 24 and 72 h of incubation. In serum-free conditions the inhibitory effect of probucol was increased fourfold after 24 h, compared to serum supplemented conditions. After 72 h, the current inhibition was similar between both culture conditions. Besides decreasing current expression, probucol shifted the GV-curve more positive combined with a shallower voltage response, changes that were more pronounced in serum-depleted conditions. The results indicated that serum-starvation had no substantial effect on the KCNQ1 + KCNE1 current in the tested CHO cells, but it amplified or accelerated the response to probucol, suggesting that halting cell proliferation is a method for enhancing the detection of delayed compound effects in HES.

Short Lysine-Containing Tripeptide as Analgesic Substance: The Possible Mechanism of Ligand-Receptor Binding to the Slow Sodium Channel.

A possible molecular mechanism of the ligand-receptor binding of Ac-Lys-Lys-Lys-NH2 (Ac-KKK-NH2) to the NaV1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH2 application at 100 nM, a significant decrease in the effective charge carried by the NaV1.8 channel activation gating system Zeff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH2 analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH2 molecule upon binding to the NaV1.8 channel are 11-12 Å, as revealed by the conformational analysis. The blind docking with the NaV1.8 channel has made it possible to locate the Ac-KKK-NH2 binding site on the extracellular side of the voltage-sensing domain VSDI. The Ac-KKK-NH2 amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Zeff value. According to the results presented, Ac-KKK-NH2 is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans.

Glyphosate impairs both structure and function of GABAergic synapses in hippocampal neurons.

Glyphosate (Gly) is a broad-spectrum herbicide responsible for the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase known to be expressed exclusively in plants and not in animals. For decades Gly has been thought to be ineffective in mammals, including humans, until it was demonstrated that rodents treated with the Gly-based herbicide Roundup showed reduced content of neurotransmitters (e.g., serotonin, dopamine, norepinephrine, and acetylcholine), increased oxidative stress in the brain associated with anxiety and depression-like behaviors and learning and memory deficits. Despite compelling evidence pointing to a neurotoxic effect of Gly, an in-depth functional description of its effects on synaptic transmission is still lacking. To investigate the synaptic alterations dependent on Gly administration we performed whole-cell patch-clamp recordings and immunocytochemistry on mouse primary cultured hippocampal neurons. Our findings reveal that 30 min incubation of Gly at the acceptable daily intake dose severely impaired inhibitory GABAergic synapses. Further analysis pointed out that Gly decreased the number of postsynaptic GABAA receptors and reduced the amplitude of evoked inhibitory postsynaptic currents, the readily releasable pool size available for synchronous release and the quantal size. Finally, a decreased number of release sites has been observed. Consistently, morphological analyses showed that the density of both pre- and post-synaptic inhibitory compartments decorating pyramidal cell dendrites was reduced by Gly. In conclusion, our experiments define for the first time the effects induced by Gly on GABAergic synapses, and reveal that Gly significantly impairs both pre- and postsynaptic mechanisms.

Cryo-EM structure of the zinc-activated channel (ZAC) in the Cys-loop receptor superfamily.

Cys-loop receptors are a large superfamily of pentameric ligand-gated ion channels with various physiological roles, especially in neurotransmission in the central nervous system. Among them, zinc-activated channel (ZAC) is a Zn2+-activated ion channel that is widely expressed in the human body and is conserved among eukaryotes. Due to its gating by extracellular Zn2+, ZAC has been considered a Zn2+ sensor, but it has undergone minimal structural and functional characterization since its molecular cloning. Among the families in the Cys-loop receptor superfamily, only the structure of ZAC has yet to be determined. Here, we determined the cryo-EM structure of ZAC in the apo state and performed structure-based mutation analyses. We identified a few residues in the extracellular domain whose mutations had a mild impact on Zn2+ sensitivity. The constriction site in the ion-conducting pore differs from the one in other Cys-loop receptor structures, and further mutational analysis identified a key residue that is important for ion selectivity. In summary, our work provides a structural framework for understanding the ion-conducting mechanism of ZAC.

Cellular and metabolic function of GIRK1 potassium channels expressed by arcuate POMC and NPY/AgRP neurons.

It is well known that the G protein-gated inwardly rectifying K+ (GIRK) channels are critical to maintain excitability of central neurons. GIRK channels consist of 4 subunits and GIRK1/GIRK2 heterotetramers are considered to be the neuronal prototype. We previously reported the metabolic significance of GIRK2 subunits expressed by the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARH). However, the role of GIRK1 subunits expressed by the neurons of ARH remains to be determined. In this study, we delineated the contribution of GIRK1 channel subunits to the excitability of the pro-opiomelanocortin (POMC) and NPY/AgRP neurons of the ARH. We further assessed the metabolic function of GIRK1 subunits expressed by these neurons. Our results provide insight into how GIRK channels regulate arcuate POMC and NPY/AgRP neurons and shape metabolic phenotypes.

[Comparative analysis of the impact of repetitive transcranial magnetic stimulation and burst transcranial magnetic stimulation at different frequencies on memory function and neuronal excitability of mice].

Transcranial magnetic stimulation (TMS) as a non-invasive neuroregulatory technique has been applied in the clinical treatment of neurological and psychiatric diseases. However, the stimulation effects and neural regulatory mechanisms of TMS with different frequencies and modes are not yet clear. This article explores the effects of different frequency repetitive transcranial magnetic stimulation (rTMS) and burst transcranial magnetic stimulation (bTMS) on memory function and neuronal excitability in mice from the perspective of neuroelectrophysiology. In this experiment, 42 Kunming mice aged 8 weeks were randomly divided into pseudo stimulation group and stimulation groups. The stimulation group included rTMS stimulation groups with different frequencies (1, 5, 10 Hz), and bTMS stimulation groups with different frequencies (1, 5, 10 Hz). Among them, the stimulation group received continuous stimulation for 14 days. After the stimulation, the mice underwent new object recognition and platform jumping experiment to test their memory ability. Subsequently, brain slice patch clamp experiment was conducted to analyze the excitability of granulosa cells in the dentate gyrus (DG) of mice. The results showed that compared with the pseudo stimulation group, high-frequency (5, 10 Hz) rTMS and bTMS could improve the memory ability and neuronal excitability of mice, while low-frequency (1 Hz) rTMS and bTMS have no significant effect. For the two stimulation modes at the same frequency, their effects on memory function and neuronal excitability of mice have no significant difference. The results of this study suggest that high-frequency TMS can improve memory function in mice by increasing the excitability of hippocampal DG granule neurons. This article provides experimental and theoretical basis for the mechanism research and clinical application of TMS in improving cognitive function.

The elevated open platform stress suppresses excitatory synaptic transmissionin the layer V anterior cingulate cortex.

There are various forms of stress including; physical, psychological and social stress. Exposure to physical stress can lead to physical sensations (e.g. hyperalgesia) and negative emotions including anxiety and depression in animals and humans. Recently, our studies in mice have shown that acute physical stress induced by the elevated open platform (EOP) can provoke long-lasting mechanical hypersensitivity. This effect appears to be related to activity in the anterior cingulate cortex (ACC) at the synaptic level. Indeed, EOP exposure induces synaptic plasticity in layer II/III pyramidal neurons from the ACC. However, it is still unclear whether or not EOP exposure alters intrinsic properties and synaptic transmission in layer V pyramidal neurons. This is essential because these neurons are known to be a primary output to subcortical structures which may ultimately impact the behavioral stress response. Here, we studied both intrinsic properties and excitatory/inhibitory synaptic transmission by using whole-cell patch-clamp method in brain slice preparations. The EOP exposure did not change intrinsic properties including resting membrane potentials and action potentials. In contrast, EOP exposure suppressed the frequency of miniature and spontaneous excitatory synaptic transmission with an alteration of kinetics of AMPA/GluK receptors. EOP exposure also reduced evoked synaptic transmission induced by electrical stimulation. Furthermore, we investigated projection-selective responses of the mediodorsal thalamus to the layer V ACC neurons. EOP exposure produced short-term depression in excitatory synaptic transmission on thalamo-ACC projections. These results suggest that the EOP stress provokes abnormal excitatory synaptic transmission in layer V pyramidal neurons of the ACC.

Nociceptive TRP channels function as molecular target for several antifungal drugs.

BACKGROUND/OBJECTIVES: Topically applied antifungal agents can induce adverse effects, such as pain and irritation. The transient receptor potential (TRP) channels-TRPA1 and TRPV1-mainly expressed in sensory neurons, act as sensors for detecting irritants. This study aims to evaluate the involvement of nociceptive channels in topical antifungal-induced pain and irritation. We tested nine topical antifungals belonging five classes: isoconazole, econazole, miconazole, clotrimazole, and ketoconazole as imidazoles; liranaftate as a thiocarbamate; terbinafine as an allylamine; amorolfine as a morpholine; and butenafine as a benzylamine. METHODS: Intracellular calcium concentrations ([Ca2+]i) and membrane currents in response to antifungals were measured to estimate channel activity using heterologously expressing cells and isolated mouse sensory neurons. RESULTS: In mouse TRPA1-expressing cells, all the tested drugs induced an increase in [Ca2+]i, which was abrogated or reduced by a TRPA1 blocker. Although many drugs evoked the TRPA1-nonspecific [Ca2+]i response at high concentrations, responses to clotrimazole, ketoconazole, and liranaftate were TRPA1 specific and elicited current responses in TRPA1-expressing cells. In mouse TRPV1-expressing cells, clotrimazole and ketoconazole elicited [Ca2+]i and current responses. In mouse sensory neurons, liranaftate-induced increase in [Ca2+]i was abrogated by a TRPA1 blocker and Trpa1 deletion. Responses to ketoconazole were inhibited by TRPA1 and TRPV1 blockers and by the genetic deletion of either channel. CONCLUSION: These results suggest that topical antifungal-induced pain and irritation are attributable to the activation of nociceptive TRPA1 and/or TRPV1 channel/s. Consequently, caution should be exercised in the use of topical antifungals with symptoms of pain.

2-BFI protects against ischemic stroke by selectively acting on NR2B-containing NMDA receptors.

BACKGROUND AND PURPOSE: The intricate roles of NMDA receptors, specifically those containing the NR2A or NR2B subunit, in ischemic stroke pathology necessitate targeted therapeutic investigations. Building on our prior discovery showcasing the neuroprotective potential of 2-(benzofuran-2-yl)-2-imidazoline (2-BFI), an imidazoline I2 receptor ligand, in inhibiting NMDA receptor currents during ischemic stroke, this study aims to elucidate the specific impact of 2-BFI on NR2A- and NR2B-containing NMDARs. EXPERIMENTAL APPROACH: Through whole-cell patch-clamp techniques, we observed an inhibition by 2-BFI on NR2A-containing NMDAR currents (IC50 = 238.6 µM) and NR2B-containing NMDAR currents (IC50 = 18.47 µM). Experiments with HEK293 cells expressing exogenous receptor subunits revealed a significantly higher affinity of 2-BFI towards NR2B-containing NMDARs. In vivo studies involved the co-administration of 2-BFI and the NR2A subunit antagonist NVP-AAM077 in rats subjected to transient middle cerebral artery occlusion (tMCAO). Key results 2-BFI exhibited a pronounced preference for inhibiting NR2B-containing NMDAR currents, leading to a notable mitigation of cerebral ischemic injury when administered in conjunction with NVP-AAM077 in the tMCAO rat model. Furthermore, alterations in the expression of downstream proteins specific to NR2B-containing NMDA receptors were observed, suggesting targeted molecular effects. Conclusion and implications This study unveils the neuroprotective potential of 2-BFI in ischemic stroke by selectively inhibiting NR2B-containing NMDA receptors. These findings lay the foundation for precise therapeutic strategies, showcasing the differential roles of NR2A and NR2B subunits and paving the way for advancements in targeted interventions for ischemic stroke treatment.

The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds.

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors-calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) - using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC50 values of 30-60 µM. DAPI and furamidine were also active against CI-AMPARs with IC50s of 50-60 µM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.

Activation of arginine vasopressin receptor 1a reduces inhibitory synaptic currents at reciprocal synapses in the mouse accessory olfactory bulb.

Central arginine vasopressin (AVP) facilitates social recognition and modulates many complex social behaviors in mammals that, in many cases, recognize each other based on olfactory and/or pheromonal signals. AVP neurons are present in the accessory olfactory bulb (AOB), which is the first relay in the vomeronasal system and has been demonstrated to be a critical site for mating-induced mate recognition (olfactory memory) in female mice. The transmission of information from the AOB to higher centers is controlled by the dendrodendritic recurrent inhibition, i.e., inhibitory postsynaptic currents (IPSCs) generated in mitral cells by recurrent dendrodendritic inhibitory inputs from granule cells. These reports suggest that AVP might play an important role in regulating dendrodendritic inhibition in the AOB. To test this hypothesis, we examined the effects of extracellularly applied AVP on synaptic responses measured from mitral and granule cells in slice preparations from 23--36-day-old Balb/c mice. To evoke dendrodendritic inhibition in a mitral cell, depolarizing voltages of -70 to 0 mV (10 ms duration) were applied to a mitral cell using a conventional whole-cell configuration. We found that AVP significantly reduced the IPSCs. The suppressive effects of AVP on the IPSCs was diminished by an antagonist for vasopressin receptor 1a (V1aR) (Manning compound), but not by an antagonist for vasopressin receptor 1b (SSR149415). An agonist for V1aRs [(Phe2)OVT] mimicked the action of AVP on IPSCs. Additionally, AVP significantly suppressed voltage-activated currents in granule cells without affecting the magnitude of the response of mitral cells to gamma-aminobutyric acid (GABA). The present results suggest that V1aRs play a role in reciprocal transmission between mitral cells and granule cells in the mouse AOB by reducing GABAergic transmission through a presynaptic mechanism in granule cells.

Force versus Response: Methods for Activating and Characterizing Mechanosensitive Ion Channels and GPCRs.

Mechanotransduction is the process whereby cells convert mechanical signals into electrochemical responses, where mechanosensitive proteins mediate this interaction. To characterize these critical proteins, numerous techniques have been developed that apply forces and measure the subsequent cellular responses. While these approaches have given insight into specific aspects of many such proteins, subsequent validation and cross-comparison between techniques remain difficult given significant variations in reported activation thresholds and responses for the same protein across different studies. Accurately determining mechanosensitivity responses for various proteins, however, is essential for understanding mechanotransduction and potential physiological implications, including therapeutics. This critical review provides an assessment of current and emerging approaches used for mechanosensitive ion channel and G-Coupled Receptors (GPCRs) stimulation and measurement, with a specific focus on the ability to quantitatively measure mechanosensitive responses.

Probing the Chemical Space of Guanidino-Carboxylic Acids to Identify the First Blockers of the Creatine-Transporter-1.

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relation for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both, inhibition of [3H]creatine uptake and transport associated currents, allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds - i.e. compound 1 (2-(N-benzylcarbamimidamido)acetic acid), and MIPA572 (=carbamimidoylphenylalanine) and MIPA573 (=carbamimidoyltryptophane) that blocked CRT-1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIP574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). Significance Statement The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1.

Electrophysiological evaluation of the effect of peptide toxins on voltage-gated ion channels: a scoping review on theoretical and methodological aspects with focus on the Central and South American experience.

The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: "electrophysiology", "patch-clamp techniques", "Ca2+ channels", "K+ channels", "cnidarian venoms", "cone snail venoms", "scorpion venoms", "spider venoms", "snake venoms", "cardiac myocytes", "dorsal root ganglia", and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel's structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins' source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering.

Membrane properties and coupling of macroglia in the optic nerve.

We established a longitudinal acute slice preparation of transgenic mouse optic nerve to characterize membrane properties and coupling of glial cells by patch-clamp and dye-filling, complemented by immunohistochemistry. Unlike in cortex or hippocampus, the majority of EGFP + cells in optic nerve of the hGFAP-EGFP transgenic mouse, a tool to identify astrocytes, were characterized by time and voltage dependent K+-currents including A-type K+-currents, properties previously described for NG2 glia. Indeed, the majority of transgene expressing cells in optic nerve were immunopositive for NG2 proteoglycan, whereas only a minority show GFAP immunoreactivity. Similar physiological properties were seen in YFP + cells from NG2-YFP transgenic mice, indicating that in optic nerve the transgene of hGFAP-EGFP animals is expressed by NG2 glia instead of astrocytes. Using Cx43kiECFP transgenic mice as another astrocyte-indicator revealed that astrocytes had passive membrane currents. Dye-filling showed that hGFAP-EGFP+ cells in optic nerve were coupled to none or few neighboring cells while hGFAP-EGFP+ cells in the cortex form large networks. Similarly, dye-filling of NG2-YFP+ and Cx43-CFP+ cells in optic nerve revealed small networks. Our work shows that identification of astrocytes in optic nerve requires distinct approaches, that the cells express membrane current patterns distinct from cortex and that they form small networks.

Old innovations and shifted paradigms in cellular neuroscience.

Once upon a time the statistics of quantal release were fashionable: "n" available vesicles (fusion sites), each with probability "p" of releasing a quantum. The story was not so simple, a nice paradigm to be abandoned. Biophysicists, experimenting with "black films," explained the astonishing rapidity of spike-induced release: calcium can trigger the fusion of lipidic vesicles with a lipid bilayer, by masking the negative charges of the membranes. The idea passed away, buried by the discovery of NSF, SNAPs, SNARE proteins and synaptotagmin, Munc, RIM, complexin. Electrophysiology used to be a field for few adepts. Then came patch clamp, and multielectrode arrays and everybody became electrophysiologists. Now, optogenetics have blossomed, and the whole field has changed again. Nice surprise for me, when Alvarez de Toledo demonstrated that release of transmitters could occur through the transient opening of a pore between the vesicle and the plasma-membrane, no collapse of the vesicle in the membrane needed: my mentor Bruno Ceccarelli had cherished this idea ("kiss and run") and tried to prove it for 20 years. The most impressive developments have probably regarded IT, computers and all their applications; machine learning, AI, and the truly spectacular innovations in brain imaging, especially functional ones, have transformed cognitive neurosciences into a new extraordinarily prolific field, and certainly let us imagine that we may finally understand what is going on in our brains. Cellular neuroscience, on the other hand, though the large public has been much less aware of the incredible amount of information the scientific community has acquired on the cellular aspects of neuronal function, may indeed help us to eventually understand the mechanistic detail of how the brain work. But this is no more in the past, this is the future.

Temperature-Dependent Activation of Thermosensitive Transient Receptor Potential Channels.

Temperature detection is essential for the survival and perpetuation of any species. Thermoreceptors in the skin sense the body temperature and also the temperatures of the ambient air and the objects. In 1997, Dr. David Julius and his colleagues found that a receptor expressed in small-diameter primary sensory neurons was activated by capsaicin (the pungent chemical in hot pepper). This receptor was also activated by temperature above 42 °C. That was the first time that a thermal receptor in primary sensory neurons has been identified. This receptor is named transient receptor potential vanilloid 1 (TRPV1). Now, 11 thermosensitive TRP channels are known. In this chapter, we summarize the reports and analyze thermosensitive TRP channels in a variety of ways to clarify the activation mechanisms by which temperature changes are sensed.