Receptor Currents Research Articles

Gating-spring stiffness increases outer-hair-cell bundle stiffness, damping, and receptor current

In our ears, outer-hair-cell bundles (OHBs) convert sound-induced forces into receptor currents that drive cochlear amplification, the process responsible for the micropascal-scale threshold and million-fold dynamic range of hearing. OHBs rely on gating springs to open mechanoelectrical-transduction (MET) ion channels, through which the receptor current flows. OHBs have larger gating-spring stiffnesses than other types of hair bundles, but we have a poor understanding of how gating-spring stiffness contributes to OHB mechanics and receptor-current regulation. Using experimentally-constrained mathematical models of the OHB, we show that the increased gating-spring stiffness in an OHB increases its stiffness and damping. The OHB’s 3D morphology reduces the contribution of gating-spring stiffness to OHB stiffness, reduces the contribution of MET-channel gating to OHB stiffness and damping, but causes additional OHB damping that rises with gating-spring stiffness. Gating-spring stiffness increases the OHB’s receptor current but decreases its displacement-current dynamic range. Strikingly, the OHB’s 3D morphology causes its force-current dynamic range to decrease with gating-spring stiffness. Our results suggest a trade-off between threshold and dynamic range regulated by OHB gating-spring stiffness.

Stereospecific Properties and Intracellular Transport of Novel Intrinsically Fluorescent Neurosteroids.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABAA) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well. We found stereospecific differences in the orientation and dynamics of the NAS as well as in their impact on membrane permeability. However, all NAS are unable to condense the lipid bilayer, in stark contrast to cholesterol. Using Förster resonance energy transfer (FRET) and electrophysiological measurements, we show that MQ-323 but not YX-11 binds at the intersubunit site of the ELICα1GABAA receptor and potentiates GABA-induced receptor currents. In aqueous solvents, YX-11 forms aggregates at much lower concentrations than MQ-323, and loading both analogs onto cyclodextrin allows for their uptake by human astrocytes, where they become enriched in lipid droplets (LDs), as shown by quantitative fluorescence microscopy. Trafficking of the NAS analogs is stereospecific, as uptake and lipid droplet targeting is more pronounced for YX-11 compared to MQ-323. In summary, we present novel minimally modified analogs of AlloP and E-AlloP, which enable us to reveal stereospecific membrane properties, allosteric receptor activation, and intracellular transport of these neurosteroids. Our fluorescence design strategy will be very useful for the analysis of other NAS in the future.

Haloperidol, a typical antipsychotic, inhibits 5-HT3 receptormediated currents in NCB-20 cells: a whole-cell patch-clamp study.

Haloperidol is a typical antipsychotic drug effective in alleviating positive symptoms of schizophrenia by blocking dopamine receptor 2 (DR2). However, it is also known to produce neuropsychiatric effects by acting on various targets other than DR. In this study, we investigated effect of haloperidol on function of 5-hydroxytryptamine (5-HT)3 receptor, a ligand-gated ion channel belonging to the serotonin receptor family using the whole-cell voltage clamp technique and NCB20 neuroblastoma cells. When co-applied with 5-HT, haloperidol inhibited 5-HT3 receptormediated currents in a concentration-dependent manner. A reduction in maximal effect (Emax) and an increase in EC50 observed during co-application indicated that haloperidol could act as a non-competitive antagonist of 5-HT3 receptors. Haloperidol inhibited the activation of 5-HT3 receptor, while also accelerating their deactivation and desensitization. The inhibitory effect of haloperidol showed no significant difference between pre- and co-application. Haloperidol did not alter the reversal potential of 5-HT3 receptor currents. Furthermore, haloperidol did not affect recovery from deactivation or desensitization of 5-HT3 receptors. It did not show a use-dependent inhibition either. These findings suggest that haloperidol can exert its inhibitory effect on 5-HT3 receptors by allosterically preventing opening of ion channels. This mechanistic insight enhances our understanding of relationships between 5-HT3 receptors and pharmacological actions of antipsychotics.

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

Background and purposeThe 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 approachThrough 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 results2-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 implicationsThis 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.

Molecular cloning, functional characterization and differential expression of two novel GABAAR-like subunits from red swamp crayfish Procambarus clarkii.

In this work, we cloned and functionally expressed two novel GABAA receptor subunits from Procambarus clarkii crayfish. These two new subunits, PcGABAA-α and PcGABAA-β2, revealed significant sequence homology with the PcGABAA-β subunit, previously identified in our laboratory. In addition, PcGABAA-α subunit also shared a significant degree of identity with the Drosophila melanogaster genes DmGRD (GABA and glycine-like receptor subunits of Drosophila) as well as PcGABAA-β2 subunit with DmLCCH3 (ligand-gated chloride channel homolog 3). Electrophysiological recordings showed that the expression in HEK cells of the novel subunits, either alone or in combination, failed to form functional homo- or heteromeric receptors. However, the co-expression of PcGABAA-α with PcGABAA-β evoked sodium- or chloride-dependent currents that accurately reproduced the time course of the GABA-evoked currents in the X-organ neurons from crayfish, suggesting that these GABA subunits combine to form two types of GABA receptors, one with cationic selectivity filter and the other preferentially permeates anions. On the other hand, PcGABAA-β2 and PcGABAA-β co-expression generated a chloride current that does not show desensitization. Muscimol reproduced the time course of GABA-evoked currents in all functional receptors, and picrotoxin blocked these currents; bicuculline did not block any of the recorded currents. Reverse transcription polymerae chain reaction (RT-PCR) amplifications and FISH revealed that PcGABAA-α and PcGABAA-β2 are predominantly expressed in the crayfish nervous system. Altogether, these findings provide the first evidence of a neural GABA-gated cationic channel in the crayfish, increasing our understanding of the role of these new GABAA receptor subunits in native heteromeric receptors.

The Role of Calcium-Dependent Desensitization in the Potentiation by Gne-9278 of NMDA Receptor Currents in Rat Cortical Neurons in vitro

The Role of Calcium-Dependent Desensitization in the Potentiation by Gne-9278 of NMDA Receptor Currents in Rat Cortical Neurons in vitro

The Ca2+-activated Cl- channel TMEM16B shapes the response time course of olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.

3D morphology of an outer-hair-cell hair bundle increases its displacement and dynamic range

In mammals, outer-hair-cell hair bundles (OHBs) transduce sound-induced forces into receptor currents and are required for the wide dynamic range and high sensitivity of hearing. OHBs differ conspicuously in morphology from other types of bundles. Here, we show that the 3D morphology of an OHB greatly impacts its mechanics and transduction. An OHB comprises rod-like stereocilia, which pivot on the surface of its sensory outer hair cell. Stereocilium pivot positions are arranged in columns and form a V shape. We measure the pivot positions and determine that OHB columns are far from parallel. To calculate the consequences of an OHB’s V shape and far-from-parallel columns, we develop a mathematical model of an OHB that relates its pivot positions, 3D morphology, mechanics, and receptor current. We find that the 3D morphology of the OHB can halve its stiffness, can double its damping coefficient, and causes stereocilium displacements driven by stimulus forces to differ substantially across the OHB. Stereocilium displacements drive the opening and closing of ion channels through which the receptor current flows. Owing to the stereocilium-displacement differences, the currents passing through the ion channels can peak versus the stimulus frequency and vary considerably across the OHB. Consequently, the receptor current peaks versus the stimulus frequency. Ultimately, the OHB’s 3D morphology can increase its receptor-current dynamic range more than twofold. Our findings imply that potential pivot-position changes owing to development, mutations, or location within the mammalian auditory organ might greatly alter OHB function.

Src dependency of the regulation of LTP by alternative splicing of GRIN1 exon 5.

Alternative splicing of Grin1 exon 5 regulates induction of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses: LTP in mice lacking the GluN1 exon 5-encoded N1 cassette (GluN1a mice) is significantly increased compared with that in mice compulsorily expressing this exon (GluN1b mice). The mechanism underlying this difference is unknown. Here, we report that blocking the non-receptor tyrosine kinase Src prevents induction of LTP in GluN1a mice but not in GluN1b. We find that activating Src enhances pharmacologically isolated synaptic N-methyl-d-aspartate receptor (NMDAR) currents in GluN1a mice but not in GluN1b. Moreover, we observe that Src activation increases the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor component of Schaffer collateral-evoked excitatory post-synaptic potentials in GluN1a mice, but this increase is prevented by blocking NMDARs. We conclude that at these synapses, NMDARs in GluN1a mice are subject to upregulation by Src that mediates induction of LTP, whereas NMDARs in GluN1b mice are not regulated by Src, leading to Src-resistance of LTP. Thus, we have uncovered that a key regulatory mechanism for synaptic potentiation is gated by differential splicing of exon 5 of Grin1. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Scorpion venom heat-resistant synthesized peptide ameliorates epileptic seizures and imparts neuroprotection in rats mediated by NMDA receptors

Finding new and effective natural products for designing antiepileptic drugs is highly important in the scientific community. The scorpion venom heat-resistant peptide (SVHRP) was purified from Buthus martensii Karsch scorpion venom, and subsequent analysis of the amino acid sequence facilitated the synthesis of a peptide known as scorpion venom heat-resistant synthesis peptide (SVHRSP) using a technique for peptide synthesis. Previous studies have demonstrated that the SVHRSP can inhibit neuroinflammation and provide neuroprotection. This study aimed to investigate the antiepileptic effect of SVHRSP on both acute and chronic kindling seizure models by inducing seizures in male rats through intraperitoneal administration of pentylenetetrazole (PTZ). Additionally, an N-methyl-D-aspartate (NMDA)-induced neuronal injury model was used to observe the anti-excitotoxic effect of SVHRSP in vitro. Our findings showed that treatment with SVHRSP effectively alleviated seizure severity, prolonged latency, and attenuated neuronal loss and glial cell activation. It also demonstrated the prevention of alterations in the expression levels of NMDA receptor subunits and phosphorylated p38 MAPK protein, as well as an improvement in spatial reference memory impairment during Morris water maze (MWM) testing in PTZ-kindled rats. In vitro experiments further revealed that SVHRSP was capable of attenuating neuronal action potential firing, inhibiting NMDA receptor currents and intracellular calcium overload, and reducing neuronal injury. These results suggest that the antiepileptic and neuroprotective effects of SVHRSP may be mediated through the regulation of NMDA receptor function and expression. This study provides new insight into therapeutic strategies for epilepsy.

Transient receptor potential vanilloid 1 involved in the analgesic effects of total flavonoids extracted from Longxuejie ().

To evaluate the analgesic effects of total flavonoids of Longxuejie (Resina Dracaenae Cochinchinensis) (TFDB) and explore the possible analgesic mechanism associated with transient receptor potential vanilloid 1 (TRPV1). Whole-cell patch clamp technique was used to observe the effects of TFDB on capsaicin-induced TRPV1 currents. Rat experiments in vivo were used to observe the analgesic effects of TFDB. Western blot and immunofluorescence experiments were used to test the change of TRPV1 expression in DRG neurons induced by TFDB. Results showed that TFDB inhibited capsaicin-induced TRPV1 receptor currents in acutely isolated dorsal root ganglion (DRG) neurons of rats and the half inhibitory concentration was (16.7 ± 1.6) mg/L. TFDB (2-20 mg/kg) showed analgesic activity in the phase Ⅱ of formalin test and (0.02-2 mg per paw) reduced capsaicin-induced licking times of rats. TFDB (20 mg/kg) was fully efficacious on complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia and capsaicin could weaken the analgesic effects. The level of TRPV1 expressions of DRG neurons was also decreased in TFDB-treated CFA-inflammatory pain rats. All these results indicated that the analgesic effect of TFDB may contribute to their modulations on both function and expression of TRPV1 channels in DRG neurons.

Evolution of Glutamate Metabolism via GLUD2 Enhances Lactate-Dependent Synaptic Plasticity and Complex Cognition.

Human evolution is characterized by rapid brain enlargement and the emergence of unique cognitive abilities. Besides its distinctive cytoarchitectural organization and extensive inter-neuronal connectivity, the human brain is also defined by high rates of synaptic, mainly glutamatergic, transmission, and energy utilization. While these adaptations' origins remain elusive, evolutionary changes occurred in synaptic glutamate metabolism in the common ancestor of humans and apes via the emergence of GLUD2, a gene encoding the human glutamate dehydrogenase 2 (hGDH2) isoenzyme. Driven by positive selection, hGDH2 became adapted to function upon intense excitatory firing, a process central to the long-term strengthening of synaptic connections. It also gained expression in brain astrocytes and cortical pyramidal neurons, including the CA1-CA3 hippocampal cells, neurons crucial to cognition. In mice transgenic for GLUD2, theta-burst-evoked long-term potentiation (LTP) is markedly enhanced in hippocampal CA3-CA1 synapses, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA receptor currents. D-lactate blocked LTP enhancement, implying that glutamate metabolism via hGDH2 potentiates L-lactate-dependent glia-neuron interaction, a process essential to memory consolidation. The transgenic (Tg) mice exhibited increased dendritic spine density/synaptogenesis in the hippocampus and improved complex cognitive functions. Hence, enhancement of neuron-glia communication, via GLUD2 evolution, likely contributed to human cognitive advancement by potentiating synaptic plasticity and inter-neuronal connectivity.

Alfaxalone is an effective anesthetic for the electrophysiological study of anoxia-tolerance mechanisms in western painted turtle pyramidal neurons.

Anoxia in the mammalian brain leads to hyper-excitability and cell death; however, this cascade of events does not occur in the anoxia-tolerant brain of the western painted turtle, Chrysemys picta belli. The painted turtle has become an important anoxia-tolerant model to study brain, heart, and liver function in the absence of oxygen, but being anoxia-tolerant likely means that decapitation alone is not a suitable method of euthanasia. Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. We find that Alfaxalone has no long-term impact on action potential parameters or whole-cell conductance. When acutely applied to naïve tissue, Alfaxalone did lengthen GABAA receptor current decay rates by 1.5-fold. Following whole-animal sedation with Alfaxalone, evoked whole cell GABAA receptor current decay rates displayed an increasing trend with 1 and 2 hours after brain sheet preparation, but showed no significant change after a 3-hour washout period. Therefore, we conclude that Alfaxalone is a suitable anesthetic for same day use in electrophysiological studies in western painted turtle brain tissue.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-d-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.Graphical

Alfaxalone does not have long-term effects on goldfish pyramidal neuron action potential properties or GABAA receptor currents.

Anesthetics have varying physiological effects, but most notably alter ion channel kinetics. Alfaxalone is a rapid induction and washout neuroactive anesthetic, which potentiates γ-aminobutyric acid (GABA)-activated GABAA receptor (GABAA-R) currents. This study aims to identify any long-term effects of alfaxalone sedation on pyramidal neuron action potential and GABAA-R properties, to determine if its impact on neuronal function can be reversed in a sufficiently short timeframe to allow for same-day electrophysiological studies in goldfish brain. The goldfish (Carassius auratus) is an anoxia-tolerant vertebrate and is a useful model to study anoxia tolerance mechanisms. The results show that alfaxalone sedation did not significantly impact action potential properties. Additionally, the acute application of alfaxalone onto naive brain slices caused the potentiation of whole-cell GABAA-R current decay time and area under the curve. Following whole-animal sedation with alfaxalone, a 3-h wash of brain slices in alfaxalone-free saline, with saline exchanged every 30 min, was required to remove any potentiating impact of alfaxalone on GABAA-R whole-cell currents. These results demonstrate that alfaxalone is an effective anesthetic for same-day electrophysiological experiments with goldfish brain slices.

MDGA2 Constrains Glutamatergic Inputs Selectively onto CA1 Pyramidal Neurons to Optimize Neural Circuits for Plasticity, Memory, and Social Behavior.

Synapse organizers are essential for the development, transmission, and plasticity of synapses. Acting as rare synapse suppressors, the MAM domain containing glycosylphosphatidylinositol anchor (MDGA) proteins contributes to synapse organization by inhibiting the formation of the synaptogenic neuroligin-neurexin complex. A previous analysis of MDGA2 mice lacking a single copy of Mdga2 revealed upregulated glutamatergic synapses and behaviors consistent with autism. However, MDGA2 is expressed in diverse cell types and is localized to both excitatory and inhibitory synapses. Differentiating the network versus cell-specific effects of MDGA2 loss-of-function requires a cell-type and brain region-selective strategy. To address this, we generated mice harboring a conditional knockout of Mdga2 restricted to CA1 pyramidal neurons. Here we report that MDGA2 suppresses the density and function of excitatory synapses selectively on pyramidal neurons in the mature hippocampus. Conditional deletion of Mdga2 in CA1 pyramidal neurons of adult mice upregulated miniature and spontaneous excitatory postsynaptic potentials, vesicular glutamate transporter 1 intensity, and neuronal excitability. These effects were limited to glutamatergic synapses as no changes were detected in miniature and spontaneous inhibitory postsynaptic potential properties or vesicular GABA transporter intensity. Functionally, evoked basal synaptic transmission and AMPAR receptor currents were enhanced at glutamatergic inputs. At a behavioral level, memory appeared to be compromised in Mdga2 cKO mice as both novel object recognition and contextual fear conditioning performance were impaired, consistent with deficits in long-term potentiation in the CA3-CA1 pathway. Social affiliation, a behavioral analog of social deficits in autism, was similarly compromised. These results demonstrate that MDGA2 confines the properties of excitatory synapses to CA1 neurons in mature hippocampal circuits, thereby optimizing this network for plasticity, cognition, and social behaviors.

Selective inhibitor of sodium-calcium exchanger, SEA0400, affects NMDA receptor currents and abolishes their calcium-dependent block by tricyclic antidepressants.

The open-channel block of N-methyl-D-aspartate receptors (NMDARs) and their calcium-dependent desensitization (CDD) represent conventional mechanisms of glutamatergic synapse regulation. In neurotrauma, neurodegeneration, and neuropathic pain the clinical benefits of cure with memantine, ketamine, Mg2+, and some tricyclic antidepressants are often attributed to NMDAR open-channel block, while possible involvement of NMDAR CDD in the therapy is not well established. Here the effects of selective high-affinity sodium-calcium exchanger (NCX) isoform 1 inhibitor, SEA0400, on NMDA-activated whole-cell currents and their block by amitriptyline, desipramine and clomipramine recorded by patch-clamp technique in cortical neurons of primary culture were studied. We demonstrated that in the presence of extracellular Ca2+, 50nM SEA0400 caused a reversible decrease of the steady-state amplitude of NMDAR currents, whereas loading neurons with BAPTA or the removal of extracellular Ca2+ abolished the effect. The decrease did not exceed 30% of the amplitude and did not depend on membrane voltage. The external Mg2+ block and 50nM SEA0400 inhibition of currents were additive, suggesting their independent modes of action. In the presence of Ca2+ SEA0400 speeded up the decay of NMDAR currents to the steady state determined by CDD. The measured IC50 value of 27nM for SEA0400-induced inhibition coincides with that for NCX1. Presumably, SEA0400 effects are induced by an enhancement of NMDAR CDD through the inhibition of Ca2+ extrusion by NCX1. SEA0400, in addition, at nanomolar concentrations could interfere with Ca2+-dependent effect of tricyclic antidepressants. In the presence of 50nM SEA0400, the IC50s for NMDAR inhibition by amitriptyline and desipramine increased by about 20 folds, as the Ca2+-dependent NMDAR inhibition disappeared. This observation highlights NCX1 involvement in amitriptyline and desipramine effects on NMDARs and unmasks competitive relationships between SEA0400 and these antidepressants. Neither amitriptyline nor desipramine could affect NCX3. The open-channel block of NMDARs by these substances was not affected by SEA0400. In agreement, SEA0400 did not change the IC50 for clomipramine, which acts as a pure NMDAR open-channel blocker. Thus, NCX seems to represent a promising molecular target to treat neurological disorders, because of the ability to modulate NMDARs by decreasing the open probability through the enhancement of their CDD.

CCK-sensitive C fibers activate NTS leptin receptor-expressing neurons via NMDA receptors.

The hormone leptin reduces food intake through actions in the peripheral and central nervous systems, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other central nervous system (CNS) sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDA receptor (NMDAR) currents, thereby potentiating vagally evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole cell recordings in brain slices taken from lepRCre × floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing, an effect mediated by CCKR1. Interestingly, NMDAR activation contributed to the current carried by spontaneous excitatory postsynaptic currents (EPSCs) and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal-type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.

Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses

Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses.

Long-lasting antiseizure effects of chronic intrasubthalamic convection-enhanced delivery of valproate

Intracerebral drug delivery is an experimental approach for the treatment of drug-resistant epilepsies that allows for pharmacological intervention in targeted brain regions. Previous studies have shown that targeted pharmacological inhibition of the subthalamic nucleus (STN) via modulators of the GABAergic system produces antiseizure effects. However, with chronic treatment, antiseizure effects are lost as tolerance develops. Here, we report that chronic intrasubthalamic microinfusion of valproate (VPA), an antiseizure medication known for its wide range of mechanisms of action, can produce long-lasting antiseizure effects over three weeks in rats. In the intravenous pentylenetetrazole seizure-threshold test, seizure thresholds were determined before and during chronic VPA application (480 μg/d, 720 μg/d, 960 μg/d) to the bilateral STN. Results indicate a dose-dependent variation in VPA-induced antiseizure effects with mean increases in seizure threshold of up to 33%, and individual increases of up to 150%. The lowest VPA dose showed a complete lack of tolerance development with long-lasting antiseizure effects. Behavioral testing with all doses revealed few, acceptable adverse effects. VPA concentrations were high in STN and low in plasma and liver. In vitro electrophysiology with bath applied VPA revealed a reduction in spontaneous firing rate, increased background membrane potential, decreased input resistance and a significant reduction in peak NMDA, but not AMPA, receptor currents in STN neurons. Our results suggest an advantage of VPA over purely GABAergic modulators in preventing tolerance development with chronic intrasubthalamic drug delivery and provide first mechanistic insights in intracerebral pharmacotherapy targeting the STN.