Electrophysiological Assays Research Articles

Abstract 13281: In vivo Reprogramming of Mouse Fibroblasts Into Cardiac Progenitor Cells for Myocardial Infarction Treatment

Introduction: Our previous study demonstrated that fibroblasts can be reprogrammed into induced cardiovascular progenitor cells (iCPCs) by a CRISPR activation approach. However, it is still unknown whether in vivo iCPC reprogramming can be achieved by direct activation of endogenous genes. Hypothesis: Direct activation of endogenous loci by using CRISPR tools targeting genomic DNA can fulfill in vivo iCPC reprogramming to repair infarcted hearts. Methods: A new fibroblast-specific CRISPRa model (Col1a2-cre/ERT;CAG-cas9) was generated. SgRNAs targeting the core promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5 were locally introduced through an AAV system in MI mice that were created by permanent LAD ligation. Heart function was measured by echocardiography and tissue sections were harvested for immunostaining. Cardiovascular cells derived from iCPCs were determined by immunostaining, ex vivo characterization, and electrophysiological assay. Results: The CRISPR system was activated in ~50% of fibroblasts after tamoxifen induction. The lineage conversion into iCPCs was identified by the Nkx2.5-eGFP reporter. eGFP+ cells were expressing proliferative markers (Ki67 and Pcna) and aggregated into spheres during suspension culture after sorting. Progenitor markers (Flk1, Ssea1, and Pdgf-α) were co-expressed in eGPF+ cells. The ejection fraction and scar formation of infarcted hearts were improved after injection of sgRNA-AAVs for 4 weeks. The engrafted iCPCs were identified by the lineage-tracing reporter and differentiated into cTnT+ cardiomyocytes, α-SMA+ smooth muscle cells, or CD31+ endothelial cells in infarcted hearts. Conclusions: Activation of endogenous loci enables in vivo iCPC reprogramming for cardiac regenerative medicine.

Hybridization of amantadine with gardenamide A enhances NMDA antagonism and in vivo anti-PD effects

Eight hybrids of amantadine (ATD) with a natural modulator gardenamide A (GA) via an alkylene carbonyl bridge or alkylene bridge have been designed and synthesized. Evaluated by electrophysiological assay, compound 5b was confirmed an enhanced NMDAR antagonist compared to ATD with IC50 value of 10.2 ± 1.2 μM. 5b has been demonstrated to reverse the damages of behavioral performance, the loss of dopaminergic neurons, the reduction of TH positive, and the increase of α-synuclein in both MPTP-treated mice and zebrafish models. In both ethological and ecological experiments, the activity of 5b was confirmed better than ATD or ATD/GA combination, and was almost equal to the positive selegiline. In vivo and in vitro, 5b is shown to reverse the ascend of NR1 and i-NOS levels. This candidate was also demonstrated the activity to down-regulated MPTP-increased Ca2+ influx in SH-SY5Y cells in a steep and sharp mode. It is displayed that 5b exerts neuroprotective effect partly by activating the PI3K/Akt signaling pathway. Taken all together, our data support that 5b is a more promising agent against PD than ATD.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

In Silico Simulations and Functional Cell Studies Evidence Similar Potency and Distinct Binding of Pacific and Caribbean Ciguatoxins

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale.Graphical

Structure–activity relationship studies in a new series of 2-amino-N-phenylacetamide inhibitors of Slack potassium channels

In this Letter we describe structure–activity relationship (SAR) studies conducted in five distinct regions of a new 2-amino-N-phenylacetamides series of Slack potassium channel inhibitors exemplified by recently disclosed high-throughput screening (HTS) hit VU0606170 (4). New analogs were screened in a thallium (Tl+) flux assay in HEK-293 cells stably expressing wild-type human (WT) Slack. Selected analogs were screened in Tl+ flux versus A934T Slack and other Slo family members Slick and Maxi-K and evaluated in whole-cell electrophysiology (EP) assays using an automated patch clamp system. Results revealed the series to have flat SAR with significant structural modifications resulting in a loss of Slack activity. More minor changes led to compounds with Slack activity and Slo family selectivity similar to the HTS hit.

Probing the structure and function of locus coeruleus projections to CNS motor centers.

The brainstem nucleus locus coeruleus (LC) sends projections to the forebrain, brainstem, cerebellum and spinal cord and is a source of the neurotransmitter norepinephrine (NE) in these areas. For more than 50 years, LC was considered to be homogeneous in structure and function such that NE would be released uniformly and act simultaneously on the cells and circuits that receive LC projections. However, recent studies have provided evidence that LC is modular in design, with segregated output channels and the potential for differential release and action of NE in its projection fields. These new findings have prompted a radical shift in our thinking about LC operations and demand revision of theoretical constructs regarding impact of the LC-NE system on behavioral outcomes in health and disease. Within this context, a major gap in our knowledge is the relationship between the LC-NE system and CNS motor control centers. While we know much about the organization of the LC-NE system with respect to sensory and cognitive circuitries and the impact of LC output on sensory guided behaviors and executive function, much less is known about the role of the LC-NE pathway in motor network operations and movement control. As a starting point for closing this gap in understanding, we propose using an intersectional recombinase-based viral-genetic strategy TrAC (Tracing Axon Collaterals) as well as established ex vivo electrophysiological assays to characterize efferent connectivity and physiological attributes of mouse LC-motor network projection neurons. The novel hypothesis to be tested is that LC cells with projections to CNS motor centers are scattered throughout the rostral-caudal extent of the nucleus but collectively display a common set of electrophysiological properties. Additionally, we expect to find these LC projection neurons maintain an organized network of axon collaterals capable of supporting selective, synchronous release of NE in motor circuitries for the purpose of coordinately regulating operations across networks that are responsible for balance and movement dynamics. Investigation of this hypothesis will advance our knowledge of the role of the LC-NE system in motor control and provide a basis for treating movement disorders resulting from disease, injury, or normal aging.

No evidence for long-range male sex pheromones in two malaria mosquitoes

Cues involved in mate seeking and recognition prevent hybridization and can be involved in speciation processes. In malaria mosquitoes, females of the two sibling species Anopheles gambiae s.s. and An. coluzzii mate in monospecific male swarms and hybrids are rare. Long-range sex pheromones driving this behaviour have been debated in literature but so far, no study has proven their existence or their absence. Here, we attempted to bring to light their existence. To put all the odds in our favour, we used different chemical ecology methods such as behavioural and electrophysiological assays as well chemical analyses, and we worked with mosquitoes at their optimal physiological mating state that is with swarming males during their natural swarming windows. Despite all our efforts, our results support the absence of long-range sex pheromones involved in swarm detection and recognition by females. We briefly discuss the implications of this finding in ecology, evolution and for control strategies.

Prenatal THC Exposure Induces Sex-Dependent Neuropsychiatric Endophenotypes in Offspring and Long-Term Disruptions in Fatty-Acid Signaling Pathways Directly in the Mesolimbic Circuitry.

Despite increased prevalence of maternal cannabis use, little is understood regarding potential long-term effects of prenatal cannabis exposure (PCE) on neurodevelopmental outcomes. While neurodevelopmental cannabis exposure increases the risk of developing affective/mood disorders in adulthood, the precise neuropathophysiological mechanisms in male and female offspring are largely unknown. Given the interconnectivity of the endocannabinoid (ECb) system and the brain’s fatty acid pathways, we hypothesized that prenatal exposure to Δ9-tetrahydrocannabinol (THC) may dysregulate fetal neurodevelopment through alterations of fatty-acid dependent synaptic and neuronal function in the mesolimbic system. To investigate this, pregnant Wistar rats were exposed to vehicle or THC (3 mg/kg) from gestational day (GD)7 until GD22. Anxiety-like, depressive-like, and reward-seeking behavior, electrophysiology, and molecular assays were performed on adult male/female offspring. Imaging of fatty acids using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was performed at prepubescence and adulthood. We report that PCE induces behavioral, neuronal, and molecular alterations in the mesolimbic system in male and female offspring, resembling neuropsychiatric endophenotypes. Additionally, PCE resulted in profound dysregulation of critical fatty acid pathways in the developing brain lipidome. Female progeny exhibited significant alterations to fatty acid levels at prepubescence but recovered from these deficits by early adulthood. In contrast, males exhibited persistent fatty acid deficits into adulthood. Moreover, both sexes maintained enduring abnormalities in glutamatergic/GABAergic function in the nucleus accumbens (NAc). These findings identify several novel long-term risks of maternal cannabis use and demonstrate for the first time, sex-related effects of maternal cannabinoid exposure directly in the developing neural lipidome.

Discovery of triazenyl triazoles as Nav1.1 channel blockers for treatment of epilepsy

The voltage-gated sodium (Nav) channel is one of most important targets for treatment of epilepsy, and rufinamide is an approved third-generation anti-seizure drug as Nav1.1 channel blocker. Herein, by triazenylation of rufinamide, we reported the triazenyl triazoles as new Nav1.1 channel blocker for treatment of epilepsy. Through the electrophysiological activity assay, compound 6a and 6e were found to modulate the inactivation voltage of Nav 1.1 channel with shift of −10.07 mv and −11.28 mV, respectively. In the pentylenetetrazole (PTZ) mouse model, 6a and 6e reduced the seizure level, prolonged seizure latency and improved the survival rate of epileptic mice at an intragastric administration of 50 mg/kg dosage. In addition, 6a also exhibited promising effectiveness in the maximal electroshock (MES) mouse model and possessed moderate pharmacokinetic profiles. These results demonstrated that 6a was a novel Nav1.1 channel blocker for treatment of epilepsy.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.

Investigating Peripheral Regional Anesthesia Using Induced Pluripotent Stem Cell Technology: Exploring Novel Terrain.

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

The T-1 conotoxin μ-SrVA from the worm hunting marine snail Conus spurius preferentially blocks the human NaV1.5 channel

Conotoxin sr5a had previously been identified in the vermivorous cone snail Conus spurius. This conotoxin is a highly hydrophobic peptide, with the sequence IINWCCLIFYQCC, which has a cysteine pattern "CC-CC" belonging to the T-1 superfamily. It is well known that this superfamily binds to molecular targets such as calcium channels, G protein-coupled receptors (GPCR), and neuronal nicotinic acetylcholine receptors (nAChR) and exerts an effect mainly in the central nervous system. However, its effects on other molecular targets are not yet defined, suggesting the potential of newly relevant molecular interactions. To find and demonstrate a potential molecular target for conotoxin sr5a electrophysiological assays were performed on three subtypes of voltage-activated sodium channels (NaV1.5, NaV1.6, and NaV1.7) expressed in HEK-293 cells with three different concentrations of sr5a(200, 400, and 600 nM). 200 nM sr5a blocked currents mediated by NaV1.5 by 33%, NaV1.6 by 14%, and NaV1.7 by 7%. The current-voltage (I-V) relationships revealed that conotoxin sr5a exhibits a preferential activity on the NaV1.5 subtype; the activation of NaV1.5 conductance was not modified by the blocking effect of sr5a, but sr5a affected the voltage-dependence of inactivation of channels. Since peptide sr5a showed a specific activity for a sodium channel subtype, we can assign a pharmacological family and rename it as conotoxin µ-SrVA.

MTOR Modulation of IKr through hERG1b-Dependent Mechanisms in Lipotoxic Heart.

In the atria, the rapid delayed rectifier channel (IKr) is a critical contributor to repolarization. In lipotoxic atria, increased activity of the serine/threonine mammalian target of rapamycin (mTOR) may remodel IKr and predispose patients to arrhythmias. To investigate whether mTOR produced defects in IKr channel function (protein expression and gating mechanisms), electrophysiology and biochemical assays in HEK293 cells stably expressing hERG1a/1b, and adult guinea pig atrial myocytes were used. Feeding with the saturated fatty acid palmitic acid high-fat diet (HFD) was used to induce lipotoxicity. Lipotoxicity-challenged HEK293 cells displayed an increased density of hERG1a/1b currents due to a targeted and significant increase in hERG1b protein expression. Furthermore, lipotoxicity significantly slowed the hERG1a/1b inactivation kinetics, while the activation and deactivation remained essentially unchanged. mTOR complex 1 (mTORC1) inhibition with rapamycin (RAP) reversed the increase in hERG1a/1b density and inactivation. Compared to lipotoxic myocytes, RAP-treated cells displayed action potential durations (APDs) and IKr densities similar to those of controls. HFD feeding triggered arrhythmogenic changes (increased the IKr density and shortened the APD) in the atria, but this was not observed in low-fat-fed controls. The data are the first to show the modulation of IKr by mTORC1, possibly through the remodeling of hERG1b, in lipotoxic atrial myocytes. These results offer mechanistic insights with implications for targeted therapeutic options for the therapy of acquired supraventricular arrhythmias in obesity and associated pathologies.

Electrophysiological Validation of Monosynaptic Connectivity between Premotor Interneurons and the aCC Motoneuron in the Drosophila Larval CNS.

The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour de force, the data remain underutilized, partly because of a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioral assays and/or GFP reconstitution across synaptic partners (GRASP) or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch-clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false-positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favorable alternative to GRASP imaging. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotor interneurons they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENT The Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed hundreds of thousands of synaptic connections of the fly larva by manual identification of anatomic landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, providing useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells and show that several genetic driver lines designed to target neurons of the larval connectome exhibit nonspecific and/or unreliable expression.

Chronic Administration of COVID-19 Drugs Fluvoxamine and Lopinavir Shortens Action Potential Duration by Inhibiting the Human Ether-à-go-go-Related Gene and Cav1.2.

Background: Old drugs for new indications in the novel coronavirus disease of 2019 (COVID-19) pandemic have raised concerns regarding cardiotoxicity, especially the development of drug-induced QT prolongation. The acute blocking of the cardiac hERG potassium channel is conventionally thought to be the primary mechanism of QT prolongation induced by COVID-19 drugs fluvoxamine (FLV) and lopinavir (LPV). The chronic impact of these medications on the hERG expression has yet to be determined. Methods: To investigate the effect of long-term incubation of FLV and LPV on the hERG channel, we used electrophysiological assays and molecular experiments, such as Western blot, RT-qPCR, and immunofluorescence, in HEK-293 cells stably expressing hERG and human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). Results: Compared to the acute effects, chronic incubation for FLV and LPV generated much lower half-maximal inhibitory concentration (IC50) values, along with a left-shifted activation curve and retarded channel activation. Inconsistent with the reduction in current, we unexpectedly found that the chronic effects of drugs promoted the maturation of hERG proteins, accompanied by the high expression of Hsp70 and low expression of Hsp90. Targeting Hsp70 using siRNA was able to reverse the effects of these drugs on hERG proteins. In addition, FLV and LPV resulted in a significant reduction of APD90 and triggered the early after-depolarizations (EADs), as well as inhibited the protein level of the L-type voltage–operated calcium channel (L-VOCC) in hiPSC-CMs. Conclusion: Chronic incubation with FLV and LPV produced more severe channel-blocking effects and contributed to altered channel gating and shortened action potential duration by inhibiting hERG and Cav1.2.

Mechanosensory pathways of scorpion pecten hair sensillae-Adjustment of body height and pecten position.

Scorpions' sensory abilities are intriguing, especially the rather enigmatic ventral comb-like chemo- and mechanosensory organs, the so-called pectines. Attached ventrally to the second mesosomal segment just posterior to the coxae of the fourth walking leg pair, the pectines consist of the lamellae, the fulcra, and a variable number of pecten teeth. The latter contain the bimodal peg sensillae, used for probing the substrate with regard to chemo- and mechanosensory cues simultaneously. In addition, the lamellae, the fulcra and the pecten teeth are equipped with pecten hair sensillae (PHS) to gather mechanosensory information. Previously, we have analyzed the neuronal pathway associated with the peg sensillae unraveling their somatotopic projection pattern in dedicated pecten neuropils. Little is known, however, regarding the projections of PHS within the scorpion nervous system. Behavioral and electrophysiological assays showed involvement of PHS in reflexive responses but how the information is integrated remains unresolved. Here, we unravel the innervation pattern of the mechanosensory pecten hair afferents in Mesobuthus eupeus and Euscorpius italicus. By using immunofluorescent labeling and injection of Neurobiotin tracer, we identify extensive arborizations of afferents, including (i) ventral neuropils, (ii) somatotopically organized multisegmental sensory tracts, (iii) contralateral branches via commissures, and (iv) direct ipsilateral innervation of walking leg neuromeres 3 and 4. Our results suggest that PHS function as sensors to elicit reflexive adjustment of body height and obstacle avoidance, mediating accurate pecten teeth alignment to guarantee functionality of pectines, which are involved in fundamental capacities like mating or navigation.

Plant-defense mimicry facilitates rapid dispersal of short-lived seeds by hornets

Rates of seed dispersal have rarely been considered important. Here, we demonstrate through field observations and experiments that rapid dispersal is essential for the unusually short-lived seeds of Aquilaria sinensis (agarwood; Thymelaeaceae), which desiccate and die within hours of exposure by fruit dehiscence in the hot, dry forest canopy in tropical southwest China. We show that three species of Vespa hornets remove most seeds within minutes of exposure. The hornets consume only the fleshy elaiosomes and deposit most seeds in damp shade, where they can germinate, a mean of 166m from the parent tree. Electrophysiological assays and field experiments demonstrate that the hornets are attracted by highly volatile short-carbon-chain (C5-C9) compounds, including aldehydes, ketones, alcohols, and acids, emitted from the dehiscent fruit capsule. These attractive fruit volatiles share 14 of 17 major electrophysiologically active compounds with those emitted from herbivore-damaged leaves, which attract predators, including hornets. Rapid seed dispersal thus appears to have been achieved in this species by the re-purposing of a rapid indirect defense mechanism. We predict that rapid seed dispersal by various mechanisms will be more widespread than currently documented and suggest that volatile attractants are more effective in facilitating this than visual signals, which are blocked by vegetation.

A massively parallel assay accurately discriminates between functionally normal and abnormal variants in a hotspot domain of KCNH2

Many genes, including KCNH2, contain "hotspot" domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this "hotspot" domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.

New TRPM8 blockers exert anticancer activity over castration-resistant prostate cancer models

TRPM8 has recently emerged as a druggable target in prostate cancer (PC) and TRPM8 modulators have been proposed as potential anticancer agents in this pathology. We have recently demonstrated their effectiveness in a castration-resistant prostate cancer (CRPC) model that is usually resistant to androgen deprivation therapy (ADT) and is considered the most aggressive form of PC. This is why the discovery of selective, effective, and potent TRPM8 modulators would improve the molecular arsenal in support of PC standard-of-care treatments. In the present paper we describe the design and the synthesis of a new series of TRPM8 antagonists, preliminarily characterized in vitro for their potency and selectivity by fluorimetric calcium assays. The preliminary screening allowed the identification of several potent (0.11 μM < IC50 < 0.49 μM) and selective compounds. The most potent derivatives were further characterized by patch-clamp electrophysiology assays, confirming their noteworthy activity. Moreover, the behavior of these compounds was investigated in 2D and 3D models of PC. These TRPM8 antagonists showed remarkable efficacy in inhibiting the growth induced by androgen in various PC cells as well as in CRPC models, confirming their potential as anticancer agents.

Catalpol Alleviates Isoflurane-Induced Hippocampal Learning and Memory Dysfunction and Neuropathological Changes in Aged Mice

Introduction: Isoflurane-associated perioperative neurocognitive disorders (PNDs) is a common complication that occurs commonly in elderly patients characterized by deterioration of hippocampus-dependent cognitive function. Mounting evidence has shown that hippocampal impairment and inflammatory processes are implicated in the pathogenesis of PNDs. Catalpol has been suggested to play a role in the modulation of neuroprotection and neurotransmission. Therefore, we surmised that catalpol may play a similar role during isoflurane-induced PNDs. Methods: In our current study, aged mice were exposed to isoflurane to develop a mouse model of PNDs and preconditioned with catalpol for 2 weeks before modeling. Three weeks after isoflurane exposure, behavioral, histological, biochemical, electrophysiological, and immunofluorescent assays were performed. Results: Our results showed that catalpol preadministration significantly alleviated cognitive impairment in the Morris water maze, novel object recognition, and Y-maze behavioral tests. Neuropathological analyses showed that catalpol preadministration reduced the loss of neurons and synapses; in line with this, it is revealed that hippocampal synaptic plasticity was restored. Mechanistically, catalpol preadministration suppressed the activation of microglia and decreased the expression of NLRP3 inflammasome. Conclusion: Our results indicate that catalpol preadministration could effectively alleviate cognitive impairment and neuropathological damage in isoflurane-exposed aged mice with its neuroprotective effects via modulation of the NLRP3 inflammatory pathway. Furthermore, the NLRP3 inflammatory pathway was revealed to be involved in these effects.