Patch-clamp Research Articles

Investigating Mechanically Activated Currents from Trigeminal Neurons of Non-Human Primates.

Pain sensation has predominantly mechanical modalities in many pain conditions. Mechanically activated (MA) ion channels on sensory neurons underly responsiveness to mechanical stimuli. The study aimed to address gaps in knowledge regarding MA current properties in higher order species such as non-human primates (NHP; common marmosets), and characterization of MA currents in trigeminal (TG) neuronal subtypes. We employed patch clamp electrophysiology and immunohistochemistry (IHC) to associate MA current types to different marmoset TG neuronal groups. TG neurons were grouped according to presumed marker expression, action potential (AP) width, characteristic AP features, after-hyperpolarization parameters, presence/absence of AP trains and transient outward currents, and responses to mechanical stimuli. Marmoset TG were clustered into 5 C-fiber and 5 A-fiber neuronal groups. The C1 group likely represent non-peptidergic C-nociceptors, the C2-C4 groups resembles peptidergic C-nociceptors, while the C5 group could be either cold-nociceptors or C-low-threshold-mechanoreceptors (C-LTMR). Among C-fiber neurons only C4 were mechanically responsive. The A1 and A2 groups are likely A-nociceptors, while the A3-A5 groups probably denote different subtypes of A-low-threshold-mechanoreceptors (A-LTMRs). Among A-fiber neurons only A1 was mechanically unresponsive. IHC data was correlated with electrophysiology results and estimates that NHP TG has ∼25% peptidergic C-nociceptors, ∼20% non-peptidergic C-nociceptors, ∼30% A-nociceptors, ∼5% C-LTMR, and ∼20% A-LTMR. Overall, marmoset TG neuronal subtypes and their associated MA currents have common and unique properties compared to previously reported data. Findings from this study could be the basis for investigation on MA current sensitizations and mechanical hypersensitivity during head and neck pain conditions.

9228 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

8388 Insulin-Like Growth Factor-1 Modulates Arcuate Kisspeptin Neuron Activity

Abstract Disclosure: J.N. Silva: None. R. Frazao: None. The kisspeptin-producing cells located at the arcuate nucleus of the hypothalamus (ARHKisspeptin) are considered the GnRH pulse generator necessary for fertility. In addition, ARHKisspeptin cells act as a metabolic sensor to control the hypothalamic-pituitary-gonadal (HPG) axis. The insulin-like growth factor-1 (IGF-1) is considered a component contributing to puberty onset and fertility. Peripherical or intracerebroventricular IGF-1 administration is sufficient to increase hypothalamic Kiss1 expression. However, whether IGF-1 acts directly on ARHKisspeptin cells to modulate the HPG axis is not known. To determine whether IGF-1 modulates ARHKisspeptin neuron activity, we used mice expressing the green fluorescent protein “humanized renilla” under the transcriptional control of the Kiss1 gene (8-10 weeks old). Fura-2 fluorescent probe was used to evaluate the state coupled or uncoupled to calcium in the face of IGF- 1 administration (0.5 μg/ml). Furthermore, whole-cell patch-clamp was employed to determine IGF-1 effects on ARHKisspeptin neuron's resting membrane potential (RMP). FURA-2 was incorporated by 64 ARHKisspeptin neurons out of 424 cells in the ARH. IGF-1 administration to the bath led to a significant increase of the intracellular calcium levels ([Ca2+]i) in 53% of ARHKisspeptin cells evaluated in male mice (34 out of 64 cells; IGF-1, 203.0 ± 26.1 nM; baseline period, 68.8 ± 9.6 nM; P< 0.0001). Non-responsive cells (30 out of 64 ARHKisspeptin neurons) exhibited average [Ca2+]i of 53.2 ± 6.3 nM after IGF-1 application vs 46.2 ± 5.6 nM in the baseline period (P= 0.3). Of note, IGF-1 also increased [Ca2+]i in 25% of non-kisspeptin cells in the ARH (93 out of 364 cells, IGF-1, 193.8 ± 12.4 nM; vs baseline period, 73.0 ± 5.6 nM, P< 0.0001). By evaluating the IGF-1 effects on ARHKisspeptin neuron’s RMP, we observed that IGF-1 tends to depolarize the RMP of 18% of cells recorded from males (4 out of 22 cells, IGF-1, -58.0 ± 3.0 mV; baseline, -64.1 ± 4.4 mV; P= 0.05), and depolarized the RMP of 44% of cells recorded from female mice (7 out of 16 cells, IGF-1, -61.1 ± 3.3 mV; baseline, -66.3 ± 2.7 mV; P= 0.002). Importantly, IGF-1-induced depolarization of ARHKisspeptin cells RMP was blocked when the recordings were performed in the presence of tetrodotoxin and ionotropic amino acid antagonists. Therefore, IGF-1-induced effects on ARHKisspeptin cells RMP depend on action potentials mediated by synaptic transmission. Our results suggest that IGF-1 indirectly modulates the activity of a large number of ARHKisspeptin neurons. Thus, situations of metabolic stress that induce changes in growth hormone secretion and, consequently, IGF-1 circulating levels, (e.g., fasting), can modulate the HPG axis through the indirect modulation of the activity of ARHKisspeptin cells. Presentation: 6/2/2024

Heterozygous KCNJ10 Variants Affecting Kir4.1 Channel Cause Paroxysmal Kinesigenic Dyskinesia.

More than 60% of paroxysmal kinesigenic dyskinesia (PKD) cases are of uncertain variants. The aim was to elucidate novel genetic contribution to PKD. A total of 476 probands with uncertain genetic causes were enrolled for whole-exome sequencing. A method of case-control analysis was applied to identify the candidate genes. Whole-cell patch-clamp recording was applied to verify the electrophysiological impact of the identified variants. A mouse model with cerebellar heterozygous knockout of the candidate gene was developed via adeno-associated virus injection, and dystonia-like phenotype inducement and rotarod tests were performed. In vivo multiunit electrical recording was applied to investigate the change in neural excitability in knockout mice. Heterozygous variants of potassium inwardly rectifying channel subfamily J member 10 (KCNJ10) clustered in PKD patients were compared with those in the control groups. Fifteen variants were detected in 16 of 522 probands (frequency = 3.07%). Patients with KCNJ10 variants tended to have a milder manifestation compared to those with PRRT2 (proline-rich transmembrane protein 2) variants. KCNJ10 variants partially altered the transmembrane location of inwardly rectifying potassium channel 4.1 (Kir4.1). The Kcnj10 expression is consistent with the natural course of PKD. Variants resulted in different degrees of reduction in cell Kir4.1 currents, and mice with heterozygous conditional knockout of Kcnj10 in the cerebellum presented dystonic posture, together with poor motor coordination and motor learning ability in rotarod tests. The firing rate of deep cerebellar nuclei was significantly elevated in Kcnj10-cKO mice. We identified heterozygous variants of KCNJ10 in PKD. Impaired function of Kir4.1 might lead to abnormal neuronal excitability, which attributed to PKD. © 2024 International Parkinson and Movement Disorder Society.

Optogenetic estimation of synaptic connections in brain slices

BackgroundDetection of synaptic connections is essential for understanding neural circuits. By using optogenetics, current injection, and glutamate uncaging to activate presynaptic cells and simultaneously recording the subsequent response of postsynaptic cells, the presence of synaptic connections can be confirmed. However, these methods present throughput challenges, such as the need for simultaneous multicellular patch-clamp recording and two-photon microscopy. These challenges lead to a trade-off between sacrificing resolution and experimental throughput. New methodWe adopted the laser, typically used for local field ablation, and combined this with post hoc analysis. We successfully approximated the synaptic connection probabilities using only an epi-fluorescence microscope and single-cell recordings. ResultsWe sequentially stimulated the channelrhodopsin 2-expressing cells surrounding the recorded cell and approximated the synaptic connection probabilities. This probability value was comparable to that obtained from simultaneous multi-cell patch-clamp recordings, which included more than 600 pairs. Comparison with existing methodsOur setup allows us to estimate connection probabilities within 100 s, outperforming existing methods. We successfully estimated synaptic connection probabilities using only the optical path typically used by an epi-fluorescence microscope and single-cell recordings. It may also be suitable for dendritic ablation experiments. ConclusionsThe proposed method simplifies the estimation of connection probabilities, which is expected to advance the study of neural circuits in conditions such as autism and schizophrenia where connection probabilities vary. Furthermore, this approach is applicable not only to local circuits but also to long-range connections, thus increasing experimental throughput.

Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential.

Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca2+ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% ΔF/F per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% ΔF/F per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca2+-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca2+ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca2+ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.

Molecular determinants of resurgent sodium currents mediated by Navβ4 peptide and A-type FHFs.

Resurgent current (INaR ) generated by voltage-gated sodium channels (VGSCs) plays an essential role in maintaining high-frequency firing of many neurons and contributes to disease pathophysiology such as epilepsy and painful disorders. Targeting INaR may present a highly promising strategy in the treatment of these diseases. Navβ4 and A-type fibroblast growth factor homologous factors (FHFs) have been identified as two classes of important INaR mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target INaR . Navβ4 and FHF4A can mediate INaR generation through the amino acid segment located in their C-terminus and N-terminus, respectively. We mainly employed site-directed mutagenesis, chimera construction and whole-cell patch-clamp recording to explore the receptor sites of Navβ4 peptide and FHF4A in Nav1.7 and Nav1.8. We show that the receptor of Navβ4-peptide involves four residues, N395, N945, F1737 and Y1744, in Nav1.7 DI-S6, DII-S6, and DIV-S6. We show that A-type FHFs generating INaR depends on the segment located at the very beginning, not at the distal end, of the FHF4 N-terminus domain. We show that the receptor site of A-type FHFs also resides in VGSC inner pore region. We further show that an asparagine at DIIS6, N891 in Nav1.8, is a major determinant of INaR generated by A-type FHFs in VGSCs. Cryo-EM structures reveal that the side chains of the critical residues project into the VGSC channel pore. Our findings provide additional evidence that Navβ4 peptide and A-type FHFs function as open-channel pore blockers and highlight channel inner pore region as a hotspot for development of novel agents targeting INaR .

Calmodulin kinase II inhibition suppresses atrioventricular conduction by regulating intracellular Ca2+ homeostasis

BackgroundCa2+/calmodulin-dependent protein kinase II (CaMKII) inhibition decelerates atrioventricular node (AVN) conduction, providing a potential treatment of tachycardia. However, the effectiveness of CaMKII inhibition on tachycardia and its underlying mechanism remains unclear. ObjectiveWe aimed to assess the effectiveness of CaMKII inhibition in reducing ventricular rates during atrial fibrillation and to elucidate the underlying mechanism in affecting AVN electrophysiology. MethodsCardiac CaMKII inhibition (AC3-I) mice were used. Transesophageal atrial pacing was performed to evaluate AVN conduction function and to induce atrial fibrillation. Patch-clamp techniques were employed to record action potentials and ionic currents in AVN cells. Intracellular Ca2+ transients and sarcomere length measurements were obtained with the IonOptix system. Masson trichrome stain was used to evaluate fibrosis in the AVN region. Western blotting and immunofluorescence techniques were employed to detect connexin expression and localization. ResultsCaMKII inhibition decreased the ventricular rate during atrial fibrillation and isoproterenol-induced tachycardia. Esophageal electrocardiogram results from AC3-I mice showed longer AVN conduction than in wild-type mice. AN- and N-type AVN cells from AC3-I mice exhibited slower action potential frequencies and diastolic depolarization rates than those of wild-type mice. The study revealed that CaMKII inhibition reduced AVN cell sarcoplasmic reticulum (SR) Ca2+ content, Ca2+ release rate from the SR during diastole, Ca2+ transient amplitude, and SR Ca2+ uptake rate. In addition, CaMKII inhibition prolonged the sarcomere diastole duration and enhanced the sensitivity of sarcomeres to Ca2+. ConclusionCaMKII inhibition effectively decreases the ventricular rate during atrial fibrillation and tachycardia by slowing down AVN conduction through suppressing Ca2+ overload in AVN cells.

Neuritin alleviates Diabetic Retinopathy by Regulating Endoplasmic Reticulum Stress in Rats.

Neuritin, a small-molecule neurotrophic factor, maintains neuronal cell activity, inhibits apoptosis, promotes process growth, and regulates neural progenitor cell differentiation, migration, and synaptic maturation. Neuritin helps retinal ganglion cells (RGCs) survive optic nerve injury in rats and regenerate axons. However, the role of Neuritin in Diabetic retinopathy (DR) is unclear. This study is intended to investigate the effect and mechanism of Neuritin in DR. For this purpose, we established DR rat models and injected Neuritin into them. This study provides a potential treatment for diabetic retinopathy. The rat model of DR was established by streptozotocin (STZ) injection, and the effect of Neuritin on DR was detected by intravitreal injection. Histological analysis was performed by H&E and TUNEL methods. The mRNA and protein expressions of endoplasmic reticulum stress (ERS) pathway-related transcription factors were detected by qRT-PCR and western blot. The blood-retinal barrier (BRB) function was assessed using the patch-clamp technique and Evans blue leakage assay. Neuritin significantly improved the retinal structure, restrained the apoptosis of retinal cells, and protected the normal function of BRB in DR model rats. Mechanistically, Neuritin may function by inhibiting the expression of GRP78, ASK1, Caspase-12, VEGF, and so on. Our results indicate that Neuritin alleviates retinal damage in DR rats via the inactive endoplasmic reticulum pathway. Our study provides a potential treatment for DR.

Regulatory role of electroacupuncture on satellite glial cell activity in the colon and dorsal root ganglion of rats with irritable bowel syndrome.

To investigate the role of satellite glial cells in irritable bowel syndrome (IBS) and the effect of electroacupuncture (EA) at the Tianshu (ST25) and Shangjuxu (ST37) combination. A model for visceral hypersensitivity in IBS was induced through colorectal distension (CRD) stimulation. Clean-grade male Sprague-Dawley (SD) rats were randomly divided into four groups: a normal group (NG), a model group (MG), an electroacupuncture group (EA), and a glial cell inhibitor group (FCA). Bilateral EA (2/100 Hz, 1 mA, 30 min) was administered at the Tianshu (ST25) and Shangjuxu (ST37) in week 6. Abdominal withdrawal reflex (AWR) scores were used to assess the behavioral response associated with visceral hyperalgesia, while hematoxylin-eosin staining was employed to evaluate pathological changes in the colon. The protein and mRNA levels of glial fibrillary acidic protein (GFAP) in the colon and colon-related dorsal root ganglion (DRG) were analyzed using immun-ofluorescence, immun-ohistochemistry, Western blotting, real-time polymerase chain reaction. The impact of EA on electrophysiological properties of colon-related DRG neurons was observed through whole-cell patch clamp analysis. EA significantly reduced the visceral pain behavior scores in rats with IBS in response to graded (20, 40, 60, 80 mm Hg) CRD stimulation. Additionally, EA downregulated the protein and mRNA expression levels of GFAP in the colon and colon-related DRG of rats with IBS. EA also regulated the resting membrane potential, rheobase and action potential of colon-related DRG neurons in rats with IBS. EA can regulate the excitatory properties of colon-related DRG neurons by downregulating the protein and mRNA expression of GFAP in the colon and colon-related DRG, indicating a potential neurobiological mechanism by which EA relieves visceral hypersensitivity in rats with IBS.

A Novel De Novo Gain-of-Function CACNA1D Variant in Neurodevelopmental Disease With Congenital Tremor, Seizures, and Hypotonia.

De novo gain-of-function variants in the CACNA1D gene, encoding the L-type voltage-gated Ca2+ channel CaV1.3, cause a multifaceted syndrome. Patients show variable degrees of autism spectrum disorder, developmental delay, epilepsy, and other neurologic and endocrine abnormalities (primary aldosteronism and/or hyperinsulinemic hypoglycemia). We study here a novel variant [c.3506G>A, NM_000720.4, p.(G1169D)] in 2 children with the same CACNA1D mutation but different disease severity. The clinical data of the study patients were collected. After molecular analysis and cloning by site-directed mutagenesis, patch-clamp recordings of transfected tsA201 cells were conducted in whole-cell configuration. The functional effects of wild-type and mutated channels were analyzed. One child is a severely affected boy with a novel de novo CACNA1D variant with additional clinical symptoms including prenatal-onset tremor, congenital respiratory insufficiency requiring continuous positive airway pressure ventilation, and sensorineural deafness. Despite episodes of hypoglycemia, insulin levels were normal. Aldosterone:renin ratios as a screening parameter for primary aldosteronism were variable. In the second patient, putative mosaicism of the p.(G1169D) variant was associated with a less severe phenotype. Patch-clamp electrophysiology of the p.(G1169D) variant in a heterologous expression system revealed pronounced activity-enhancing gating changes, including a shift of channel activation and inactivation to more hyperpolarized potentials, as well as impaired channel inactivation and deactivation. Despite retained sensitivity to the Ca2+ channel blocker isradipine in vitro, no beneficial effects of isradipine or nifedipine treatment were observed in the index case. Through this report, we expand the knowledge about the disease presentation in patients with CACNA1D variants and show the novel variant's modulatory effects on CaV1.3 gating.

Isoflurane anesthesia decreases excitability of inhibitory neurons in the basolateral amygdala leading to anxiety‑like behavior in aged mice.

Anxiety after surgery can be a major factor leading to postoperative cognitive dysfunction, particularly in elderly patients. The role of inhibitory neurons in the basolateral amygdala (BLA) in anxiety-like behaviors in aged mice following isoflurane anesthesia remains unclear. Therefore, the present study aimed to investigate the role of inhibitory neurons in isoflurane-treated mice. A total of 30 C57BL/6 mice (age, 13 months) were allocated into the control and isoflurane anesthesia groups (15 mice/group) and were then subjected to several neurological assessments. Behavioral testing using an elevated plus maze test showed that aged mice in the isoflurane anesthesia group displayed significant anxiety-like behavior, since they spent more time in the closed arm, exhibited more wall climbing behavior and covered more distance. In addition, whole-cell patch-clamp recording revealed that the excitability of the BLA excitatory neurons was notably increased following mice anesthesia with isoflurane, while that of inhibitory neurons was markedly reduced. Following mice treatment with diazepam, the excitability of the BLA inhibitory neurons was notably increased compared with that of the excitatory neurons, which was significantly attenuated. Overall, the results of the current study indicated that anxiety-like behavior could occur in aged mice after isoflurane anesthesia, which could be caused by a reduced excitability of the inhibitory neurons in the BLA area. This process could enhance excitatory neuronal activity in aged mice, thus ultimately promoting the onset of anxiety-like behaviors.

Characterization of a novel variant in KCNJ16, encoding Kir5.1 channel.

The essential role of the inwardly rectifying potassium channel Kir5.1 (KCNJ16) in controlling electrolyte homeostasis and blood pressure has been demonstrated in human and animal studies. Previous studies have identified several bi-allelic mutations of KCNJ16 in humans, causing severe hypokalemia, renal salt wasting, and disturbed acid-base homeostasis. Here, we identified a novel homozygous variant of KCNJ16, I26T, in an Amish patient affected with polydipsia, developmental delay, and chronic metabolic acidosis with low serum bicarbonate concentration. Subsequently, we generated the rat model with I26T mutation using Dahl salt-sensitive rat (I26T rat) to characterize this variant. The male mutant rats displayed similar blood pressure and electrolyte homeostasis under baseline and with a high salt (4% NaCl) challenge. Blood pH, HCO3 - and renal damage also remained similar between WT and I26T rats after high salt challenge. Additionally, single-channel patch clamp analysis revealed similar channel activity in CHO cells overexpressed with WT and I26T mutant Kir4.1/5.1 channels. In summary, this study reported a novel variant in KCNJ16, namely I26T, which is likely a benign variant and not associated with pathologic phenotype in either human or Dahl salt-sensitive rats, indicating that the type/location of variant should be considered when diagnosing and treating patients with KCNJ16 mutations.

Robotic Patch Clamp Electrophysiological Signal Measurement in Multiple Brain Regions Based on Brain Slice Registration

Robotic Patch Clamp Electrophysiological Signal Measurement in Multiple Brain Regions Based on Brain Slice Registration

The Need for Speed; Investigating Channelopathy-Associated Epilepsy Using High Throughput Electrophysiological Approaches.

Pathogenic variants in genes encoding ion channels are frequently discovered in monogenic disorders associated with epilepsy and neurodevelopmental disorders. This review covers advances in the use of automated patch clamp recording for determining the functional consequences of epilepsy-associated ion channel variants and the use of induced pluripotent stem cell (iPSC) derived neurons for in-depth investigations of the physiological consequences of such variants. The combination of these advanced technologies was a focus of the recently completed NINDS-funded Channelopathy-associated Epilepsy Research Center without Walls.

Deregulation of Melatonin Receptors and Differential Modulation of After-Hyperpolarization and Ih Currents Using Melatonin Treatment Due to Amyloid-β-Induced Neurotoxicity in the Hippocampus.

Treatment with melatonin is routinely prescribed for its potent antioxidant and cognitive-promoting effects, nevertheless, it has yet to find neuromodulatory effects in normal and disease conditions. Therefore, to investigate its neuromodulatory mechanisms, melatonin was systemically administered over 10 consecutive days to both intracortical normal saline- and amyloid-β 1-42 (Aβ) peptide-injected rats. At the behavioral level, treatment with melatonin was associated with reduced efficacy in restoring Aβ-induced deficit in passive-avoidance memory. Whole-cell patch-clamp recordings from CA1 pyramidal neurons revealed that melatonin treatment reduced spontaneous and evoked intrinsic excitability in control rats while exerting a reduction of spontaneous, but not evoked activity, in the Aβ-injected group. Interestingly, treatment with melatonin enhances after-hyperpolarization in control, but not Aβ-injected rats. In contrast, our voltage-clamp study showed that Ih current is significantly enhanced by Aβ injection, and this effect is further strengthened by treatment with melatonin in Aβ-injected rats. Finally, we discovered that the transcription of melatonin receptors 1 (MT1) and 2 (MT2) is significantly upregulated in the hippocampi of Aβ-injected rats. Collectively, our study demonstrates that systemic treatment with melatonin has differential neuromodulation on CA1 neuronal excitability, at least in part, via differential effects on after-hyperpolarization and Ih currents due to Aβ-induced neurotoxicity.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice.

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

Early life stress modulates neonatal somatosensation and the transcriptional profile of immature sensory neurons

Abstract Early life stress (ELS) is associated with an increased risk of experiencing chronic pain during adulthood, but surprisingly little is known about the short-term influence of ELS on nociceptive processing in the immature nervous system and the concomitant effects on somatosensation in the neonate. Here, we investigate how ELS modulates pain in neonatal mice and the transcriptional and electrophysiological signatures of immature dorsal root ganglia (DRG). Shortly after the administration of a neonatal limiting bedding (NLB) paradigm from postnatal days (P)2 to P9, both male and female pups exhibited robust hypersensitivity in response to tactile, pressure, and noxious cold stimuli compared with a control group housed under standard conditions, with no change in their sensitivity to noxious heat. Bulk RNA-seq analysis of L3-L5 DRGs at P9 revealed significant alterations in the transcription of pain- and itch-related genes following ELS, highlighted by a marked downregulation in Sst, Nppb, Chrna6, Trpa1, and Il31ra. Nonetheless, ex vivo whole-cell patch-clamp recordings from putative A- and C-fiber sensory neurons in the neonatal DRG found no significant changes in their intrinsic membrane excitability following NLB. Overall, these findings suggest that ELS triggers hyperalgesia in neonates across multiple pain modalities that is accompanied by transcriptional plasticity within developing sensory neurons. A better understanding of the mechanisms governing the interactions between chronic stress and pain during the neonatal period could inform the future development of novel interventional strategies to relieve pain in infants and children who have experienced trauma.

Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA initiative

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC50 for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC50 for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.