Electrophysiological Techniques Research Articles

Low-Power Fully Integrated 256-Channel Nanowire Electrode-on-Chip Neural Interface for Intracellular Electrophysiology.

Intracellular electrophysiology, a vital and versatile technique in cellular neuroscience, is typically conducted using the patch-clamp method. Despite its effectiveness, this method poses challenges due to its complexity and low throughput. The pursuit of multi-channel parallel neural intracellular recording has been a long-standing goal, yet achieving reliable and consistent scaling has been elusive because of several technological barriers. In this work, we introduce a micropower integrated circuit, optimized for scalable, high-throughput in vitro intrinsically intracellular electrophysiology. This system is capable of simultaneous recording and stimulation, implementing all essential functions such as signal amplification, acquisition, and control, with a direct interface to electrodes integrated on the chip. The electrophysiology system-on-chip (eSoC), fabricated in 180nm CMOS, measures 2.236 mm × 2.236 mm. It contains four 8 × 8 arrays of nanowire electrodes, each with a 50 μm pitch, placed over the top-metal layer on the chip surface, totaling 256 channels. Each channel has a power consumption of 0.47 μW, suitable for current stimulation and voltage recording, and covers 80 dB adjustable range at a sampling rate of 25 kHz. Experimental recordings with the eSoC from cultured neurons in vitro validate its functionality in accurately resolving chemically induced multi-unit intracellular electrical activity.

Sex differences in motor learning flexibility are accompanied by sex differences in mushroom spine pruning of the mouse primary motor cortex during adolescence

BackgroundAlthough males excel at motor tasks requiring strength, females exhibit greater motor learning flexibility. Cognitive flexibility is associated with low baseline mushroom spine densities achieved by pruning which can be triggered by α4βδ GABAA receptors (GABARs); defective synaptic pruning impairs this process.MethodsWe investigated sex differences in adolescent pruning of mushroom spine pruning of layer 5 pyramidal cells of primary motor cortex (L5M1), a site essential for motor learning, using microscopic evaluation of Golgi stained sections. We assessed α4GABAR expression using immunohistochemical and electrophysiological techniques (whole cell patch clamp responses to 100 nM gaboxadol, selective for α4βδ GABARs). We then compared performance of groups with different post-pubertal mushroom spine densities on motor learning (constant speed) and learning flexibility (accelerating speed following constant speed) rotarod tasks.ResultsMushroom spines in proximal L5M1 of female mice decreased >60% from PND35 (puberty onset) to PND56 (Pubertal: 2.23 ± 0.21 spines/10 μm; post-pubertal: 0.81 ± 0.14 spines/10 μm, P < 0.001); male mushroom spine density was unchanged. This was due to greater α4βδ GABAR expression in the female (P < 0.0001) because α4 -/- mice did not exhibit mushroom spine pruning. Although motor learning was similar for all groups, only female wild-type mice (low mushroom spine density) learned the accelerating rotarod task after the constant speed task (P = 0.006), a measure of motor learning flexibility.ConclusionsThese results suggest that optimal motor learning flexibility of female mice is associated with low baseline levels of post-pubertal mushroom spine density in L5M1 compared to male and female α4 -/- mice.

Oral Administration of a Specific p300/CBP Lysine Acetyltransferase Activator Induces Synaptic Plasticity and Repairs Spinal Cord Injury.

TTK21 is a small-molecule activator of p300/creb binding protein (CBP) acetyltransferase activity, which, upon conjugation with a glucose-derived carbon nanosphere (CSP), can efficiently cross the blood-brain barrier and activate histone acetylation in the brain. Its role in adult neurogenesis and retention of long-term spatial memory following intraperitoneal (IP) administration is well established. In this study, we successfully demonstrate that CSP-TTK21 can be effectively administered via oral gavage. Using a combination of molecular biology, microscopy, and electrophysiological techniques, we systematically investigate the comparative efficacy of oral administration of CSP and CSP-TTK21 in wild-type mice and evaluate their functional effects in comparison to intraperitoneal (IP) administration. Our findings indicate that CSP-TTK21, when administered orally, induces long-term potentiation in the hippocampus without significantly altering basal synaptic transmission, a response comparable to that achieved through IP injection. Remarkably, in a spinal cord injury model, oral administration of CSP-TTK21 exhibits efficacy equivalent to that of IP administration. Furthermore, our research demonstrates that oral delivery of CSP-TTK21 leads to improvements in motor function, histone acetylation dynamics, and increased expression of regeneration-associated genes (RAGs) in a spinal injury rat model, mirroring the effectiveness of IP administration. Importantly, no toxic and mutagenic effects of CSP-TTK21 are observed at a maximum tolerated dose of 1 g/kg in Sprague-Dawley (SD) rats via the oral route. Collectively, these results underscore the potential utility of CSP as an oral drug delivery system, particularly for targeting the neural system.

The Novel Long QT Syndrome Type 2-associated F129I Mutation in the KCNH2 Gene Significantly Affects I Kr Through the hERG1 Homomeric and Heteromeric Potassium Channels

Objective: The long QT syndrome type 2 is caused by the loss-of-function mutations in the KCNH2 gene, which encodes hERG1, the voltage-gated potassium channel. The hERG1 channels conduct rapid delayed rectifier K+ currents (I Kr) in the human cardiac tissue. KCNH2 encodes 2 main isoforms—hERG1a and hERG1b, which assemble to form the homomeric or heteromeric hERG1 channels. However, the functional characteristics of the heteromeric hERG1 channels in long QT syndrome type 2 are not clear. In this study, a novel mutation in the N-terminus of hERG1a (F129I) was identified in a proband of long QT syndrome type 2. The purpose of this study was to identify the electrophysiological change of homomeric and heteromeric hERG1 channels with the F129I-hERG1a. Methods: Candidate genes were screened by direct sequencing. F129I-hERG1a was cloned in the pcDNA3.1 vector by site-directed mutagenesis. Then, the wild-type (WT) hERG1a and/or F129I-hERG1a were transiently expressed in the HEK293 cells with or without hERG1b co-expression. The expression levels of the transgenes, cellular distribution of hERG1a and hERG1b, and the electrophysiological features of the homomeric and the heteromeric hERG1 channels with the WT-hERG1a or F129I-hERG1a were analyzed using whole-cell patch-clamp electrophysiology, western blotting, and immunofluorescence techniques. Results: The proband was clinically diagnosed with long QT syndrome type 2 and carried a heterozygous mutation c.385T>A (F129I) in the KCNH2 gene. Electrophysiology study proved that the F129I substitution in hERG1a significantly decreased I Kr in both the homomeric and heteromeric hERG1channels by 86% and 70%, respectively (WT-hERG1a (54.88 ± 18.74) pA/pF vs. F129I-hERG1a (7.34 ± 1.90) pA/pF, P < 0.001; WT-hERG1a/hERG1b (89.92 ± 24.51) pA/pF vs. F129I-hERG1a/hERG1b (26.54 ± 9.83) pA/pF, P < 0.001). The voltage dependence of IKr activation (V½ and k) was not affected by the mutation in both the homomeric and heteromeric hERG1 channels. The peak current densities and the kinetic characteristics of IKr were comparable for both WT/F129I-hERG1a and WT-hERG1a. The channel inactivation and deactivation analysis showed that F129I substitution did not affect deactivation of the homomeric hERG1a channel, but significantly accelerated the deactivation and recovery from inactivation of the heteromeric hERG1a/hERG1b channel based on the time constants of fast and slow recovery from deactivation F129I-hERG1a/hERG1b vs. WT-hERG1a/hERG1b (P < 0.05). Western blotting and immunofluorescence labeling experiments showed that maturation and intracellular trafficking of the F129I-hERG1a protein was impaired and potentially increased the ratio of hERG1b to hERG1a in the F129I-hERG1a/hERG1b tetramer channel, thereby resulting in electrophysiological changes characteristic of the long QT syndrome type 2 pathology. Conclusions: I Kr was significantly reduced in the homomeric and heteromeric hERG1 channels with F129I-hERG1a. The F129I mutation significantly accelerated the deactivation and recovery from inactivation of the heteromeric F129I-hERG1a/hERG1b channel. F129I-hERG1a exhibited impaired maturation and intracellular trafficking, thereby potentially increasing the ratio of the hERG1b to hERG1a stoichiometry in the hERG1 tetrameric channel. These changes demonstrated the importance of the heteromeric hERG1 channel in long QT syndrome type 2 pathophysiology.

P.062 Normal NCS in 42-year-old man with PMP22 duplication

Background: Charcot Marie Tooth disease is a polygenic disorder with cannonical features of distal amyotrophy, acrohypesthesia, and tight tendoachilles of either axonal (type 2) or demeylinating (type 1) varieties. Type 1 CMT patients are required to possess conduction slowing of a sufficient degree to qualify as demyelinating. Presented is a middle-aged man with an unremarkable neurologic exam and normal electrophysiology. Methods: Standard electrophysiological techniques were employed to obtain the nerve conduction data (Natus Nicolet EDX AT2; Middleton, WI, USA). Repeated next generation sequencing and deletion/duplication analyses were performed. Results: The nerve conduction studies showed no evidence of demyelination in the upper or lower extremeties. The duplication error was confirmed with repeat testing. The heterozygous PMP22 gene duplication encompassed the entire coding sequence involving exons 1-5. Conclusions: CMT1A accounts for the vast majority of dysmyelinating hereditary neuropathies. Phenotypic variability is well described. Presentations include (a) classic conduction slowing, (b) intermediate slowing, (c) conduction block, (d) HNPP-like, (e) absent CMAPs, and (f) normal NCSs in young infants. This is the first case of a neurologically intact adult with CMT1A. Cryptogenic genetic modifier-effect(s) are posited as a possible explanation of the lack of penetrance. Identifying the nature of this modification may prove instructive for future therapies.

Electrophysiological Mechanisms and Validation of Ferritin-Based Magnetogenetics for Remote Control of Neurons.

Magnetogenetics was developed to remotely control genetically targeted neurons. A variant of magnetogenetics uses magnetic fields to activate transient receptor potential vanilloid (TRPV) channels when coupled with ferritin. Stimulation with static or radiofrequency (RF) magnetic fields of neurons expressing these channels induces Ca2+ transients and modulates behavior. However, the validity of ferritin-based magnetogenetics has been questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Here, we validated the magnetogenetic approach FeRIC using electrophysiological and imaging techniques. Previously, interference from RF stimulation rendered patch-clamp recordings inaccessible for magnetogenetics. We solved this limitation for FeRIC, and we studied the bioelectrical properties of neurons expressing TRPV4 (non-selective cation channel) and TMEM16A (chloride permeable channel) coupled to ferritin (FeRIC channels) under RF stimulation. We used cultured neurons obtained from rat hippocampus of either sex. We show that RF decreases the membrane resistance and depolarizes the membrane potential in neurons expressing TRPV4FeRIC RF does not directly trigger action potential firing but increases the neuronal basal spiking frequency. In neurons expressing TMEM16AFeRIC, RF decreases the membrane resistance, hyperpolarizes the membrane potential, and decreases the spiking frequency. Additionally, we corroborated the previously described biochemical mechanism responsible for RF-induced activation of ferritin-coupled ion channels. We solved an enduring problem for ferritin-based magnetogenetics, obtaining direct electrophysiological evidence of RF-induced activation of ferritin-coupled ion channels. We found that RF does not yield instantaneous changes in neuronal membrane potentials. Instead, RF produces responses that are long-lasting and moderate, but effective in controlling the bioelectrical properties of neurons.Significance statement Cell-specific and non-invasive stimulation can be a powerful tool for modulating neuronal circuits and functions. Magnetogenetic techniques that are fully genetically encoded provide such tools. However, there have been significant controversies surrounding the efficacy and underlying mechanisms of magnetogenetics. Here, we demonstrate that by employing a fully genetically encoded magnetogenetic approach called FeRIC, we can modulate neuronal voltage, inducing either depolarization or hyperpolarization through the activation of ion channels with magnetic fields; we validate this modulation mechanism with the gold-standard patch-clamp technique. We further discover that this neuronal modulation is not achieved by instantaneously triggering action potentials as previously assumed, but by modulating neuronal excitability.

Antibiotic Loaded Phytosomes as a Way to Develop Innovative Lipid Formulations of Polyene Macrolides.

The threat of antibiotic resistance of fungal pathogens and the high toxicity of the most effective drugs, polyene macrolides, force us to look for new ways to develop innovative antifungal formulations. The aim of this study was to determine how the sterol, phospholipid, and flavonoid composition of liposomal forms of polyene antibiotics, and in particular, amphotericin B (AmB), affects their ability to increase the permeability of lipid bilayers that mimic the membranes of mammalian and fungal cells. To monitor the membrane permeability induced by various polyene-based lipid formulations, a calcein leakage assay and the electrophysiological technique based on planar lipid bilayers were used. The replacement of cholesterol with its biosynthetic precursor, 7-dehydrocholesterol, led to a decrease in the ability of AmB-loaded liposomes to permeabilize lipid bilayers mimicking mammalian cell membranes. The inclusion of plant flavonoid phloretin in AmB-loaded liposomes increased the ability of the formulation to disengage a fluorescent marker from lipid vesicles mimicking the membranes of target fungi. I-V characteristics of the fungal-like lipid bilayers treated with the AmB phytosomes were symmetric, demonstrating the functioning of double-length AmB pores and assuming a decrease in the antibiotic threshold concentration. The therapeutic window of polyene lipid formulations might be expanded by varying their sterol composition. Polyene-loaded phytosomes might be considered as the prototypes for innovative lipid antibiotic formulations.

Benzaldehyde acts as a behaviorally active component in brewer's yeast protein powder which attracts B. dorsalis through olfaction.

The Oriental fruit fly, Bactrocera dorsalis, is a significant pest that damages a variety of fruit crops. The effectiveness of chemical pesticides against such pests is limited, raising concerns about pesticide residues and resistance. Proteins naturally attract B. dorsalis and have led to the development of a management strategy known as protein bait attractant technology (BAT). Although the attraction of protein sources to B. dorsalis is well-documented, the biologically active components within these sources are not fully understood. This study employed analytical chemistry, behavioral tests, and electrophysiological techniques to investigate the behaviorally active components of beer yeast protein powder (BYPD), aiming to provide a basis for improving and developing protein baits. An olfactory trap assay confirmed the attractiveness of BYPD, and five components with high abundance were identified from its headspace volatiles using GC-MS. These components included ethanol, isoamyl alcohol, ethyl decanoate, benzaldehyde, and phenylethyl alcohol. Mixtures of these five components demonstrated significant attraction to B. dorsalis adults, with benzaldehyde identified as a potential key component. The attractiveness of benzaldehyde required a relatively large dose, and it was most attractive to adults that had been starved from dusk until the following morning. Attraction of adult flies to benzaldehyde appeared mainly mediated by inputs from olfactory receptors. While EAG data supports that ionotropic receptors could influence the detection of benzaldehyde in female adults, they did not affect female behavior towards benzaldehyde. These findings indicate that benzaldehyde is an important behaviorally active component in BYPD and offer insights for developing novel protein lures to control B. dorsalis in an environmentally friendly manner.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Targeting the adenosinergic system in restless legs syndrome: A pilot, "proof-of-concept" placebo-controlled TMS-based protocol.

Restless Legs Syndrome (RLS) is a common sleep disorder characterized by an urge to move the legs that is responsive to movement (particularly during rest), periodic leg movements during sleep, and hyperarousal. Recent evidence suggests that the involvement of the adenosine system may establish a connection between dopamine and glutamate dysfunction in RLS. Transcranial magnetic stimulation (TMS) is a non-invasive electrophysiological technique widely applied to explore brain electrophysiology and neurochemistry under different experimental conditions. In this pilot study protocol, we aim to investigate the effects of dipyridamole (a well-known enhancer of adenosinergic transmission) and caffeine (an adenosine receptor antagonist) on measures of cortical excitation and inhibition in response to TMS in patients with primary RLS. Initially, we will assess cortical excitability using both single- and paired-pulse TMS in patients with RLS. Then, based on the measures obtained, we will explore the effects of dipyridamole and caffeine, in comparison to placebo, on various TMS parameters related to cortical excitation and inhibition. Finally, we will evaluate the psycho-cognitive performance of RLS patients to screen them for cognitive impairment and/or mood-behavioral dysfunction, thus aiming to correlate psycho-cognitive findings with TMS data. Overall, this study protocol will be the first to shed lights on the neurophysiological mechanisms of RLS involving the modulation of the adenosine system, thus potentially providing a foundation for innovative "pharmaco-TMS"-based treatments. The distinctive TMS profile observed in RLS holds indeed the potential utility for both diagnosis and treatment, as well as for patient monitoring. As such, it can be considered a target for both novel pharmacological (i.e., drug) and non-pharmacological (e.g., neuromodulatory), "TMS-guided", interventions.

Morphological Tracing and Functional Identification of Monosynaptic Connections in the Brain: A Comprehensive Guide.

Behavioral studies play a crucial role in unraveling the mechanisms underlying brain function. Recent advances in optogenetics, neuronal typing and labeling, and circuit tracing have facilitated the dissection of the neural circuitry involved in various important behaviors. The identification of monosynaptic connections, both upstream and downstream of specific neurons, serves as the foundation for understanding complex neural circuits and studying behavioral mechanisms. However, the practical implementation and mechanistic understanding of monosynaptic connection tracing techniques and functional identification remain challenging, particularly for inexperienced researchers. Improper application of these methods and misinterpretation of results can impede experimental progress and lead to erroneous conclusions. In this paper, we present a comprehensive description of the principles, specific operational details, and key steps involved in tracing anterograde and retrograde monosynaptic connections. We outline the process of functionally identifying monosynaptic connections through the integration of optogenetics and electrophysiological techniques, providing practical guidance for researchers.

B1AR regulation of the Cav1.2 via the SAP97 complex

Beta adrenergic (BAR) signaling is stimulated by the catecholamines norepinephrine (NE) and epinephrine (EPI) to control cardiac contractility. The beta-1-AR (B1AR) is the major cardiac subtype. B1AR signals canonically through the Gas subunit, leading to increases in cAMP and PKA; however, it can also signal via the Gai subunit, leading to increased NOS1, cGMP, and PKG levels. B1AR-NOS1 signaling leads to increased contractility and calcium cycling in cardiomyocytes. We found a novel complex within cardiomyocytes in which the B1AR, Cav1.2, and NOS1 proteins are held in proximity by the membrane scaffolding protein SAP97. In heart failure, there is a decrease in SAP97 expression and impaired B1AR signaling. Cardiac-specific deletion of SAP97 disrupts this complex. In the SAP97-cKO mice, there are minimal changes in B1AR-Gas; however, B1AR-Gai signaling is decreased, with a loss of B1AR-NOS1 signaling. Furthermore, we see a decrease in whole cell calcium currents and cardiac contractility. In mice lacking the NOS1, we see the same decrease in calcium currents. We hypothesize SAP97 is necessary to maintain B1AR modulation of Cav1.2 in cardiomyocytes. Using electrophysiology and biochemical techniques, we aim to elucidate the role of SAP97 in B1AR modulation of Cav1.2 and how this is impacted by the impairment of B1AR-Gai signaling. T32HL086350 RO1-HL147263. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Retromer- and Retriever-Associated Sorting Nexins Regulate Traffcking of KCa2.3 Channels

Correct function of the Ca2+-activated K+ channel KCa2.3 (SK3) is essential for several physiological processes. Activity of KCa2.3 in neurons modulates the rate of action potential firing; in endothelial cells activation of KCa2.3 channels induces vasodilation, and accordingly, a decrease in blood pressure. KCa2.3 activity (i.e. currents) can be modulated by two main variables: the open probability ( Po) and the number of channels inserted into the plasma membrane ( N). Numerous pharmacological compounds have been identified and developed to alter KCa2.3 Po. In contrast, the mechanisms that control delivery of KCa2.3 to and from the plasma membrane are still poorly understood. After endocytosis, plasma membrane proteins are targeted to the endosomal network which is a key area for sorting membrane-bound proteins. Several endosomal-associated coat complexes have been identified that ‘rescue’ proteins from degradation and sequester proteins into recycling tubules for transport back to the plasma membrane. These complexes include Retromer, Retriever, and Commander. Associated with these complexes are members of the Sorting Nexin (SNX) family. SNX proteins bind to specific amino acid motifs to sort proteins for Retromer- or Retriever-mediated recycling. Interestingly, the Devor laboratory has demonstrated KCa2.3 can be rapidly recycled back into the plasma membrane after endocytosis. Based on those data, we hypothesised that the Retromer and Retriever complexes regulate KCa2.3 recycling. To investigate this, we are using biochemical [e.g. cell surface biotinylation, co-immunoprecipitation (co-IP)] and electrophysiological (e.g. patch-clamp) techniques. First, we determined the role of SNXs in KCa2.3 traffcking by using siRNA to knock down SNX protein expression in Fisher rat thyroid (FRT) cells expressing heterologous KCa2.3. Knockdown of Retromer-associated SNX3 or Retriever-associated SNX17 significantly decreased the cell surface population of KCa2.3 ( P<0.01, n=4, each). With protein-protein interaction experiments, KCa2.3 co-IPed with SNX17 and SNX3, but not Retromer-associated SNX27 ( n=3, each). Electrophysiology experiments are underway to determine if the knockdown of SNXs alters the functional expression of KCa2.3. and We are also investigating if KCa2.3 traffcking in primary endothelial cells requires SNX3 and SNX17. These results suggest that SNX3 and SNX17 are novel regulators of KCa2.3 traffcking. This work was supported by grants from NIH (HL083060, HL092157) and the Cystic Fibrosis Foundation (DEVOR20GO) to D.C.D.; Lottery Health New Zealand (R-LHR-2019-101706) to F.J.M.; and Aim Fund grant to K.L.H., and the Department of Physiology, University of Otago. M.J.E.L. was supported by a doctoral scholarship from the University of Otago. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structural, Functional, and Genetic Changes Surrounding Electrodes Implanted in the Brain.

Implantable neurotechnology enables monitoring and stimulating of the brain signals responsible for performing cognitive, motor, and sensory tasks. Electrode arrays implanted in the brain are increasingly used in the clinic to treat a variety of sources of neurological diseases and injuries. However, the implantation of a foreign body typically initiates a tissue response characterized by physical disruption of vasculature and the neuropil as well as the initiation of inflammation and the induction of reactive glial states. Likewise, electrical stimulation can induce damage to the surrounding tissue depending on the intensity and waveform parameters of the applied stimulus. These phenomena, in turn, are likely influenced by the surface chemistry and characteristics of the materials employed, but further information is needed to effectively link the biological responses observed to specific aspects of device design. In order to inform improved design of implantable neurotechnology, we are investigating the basic science principles governing device-tissue integration. We are employing multiple techniques to characterize the structural, functional, and genetic changes that occur in the cells surrounding implanted electrodes. First, we have developed a new "device-in-slice" technique to capture chronically implanted electrodes within thick slices of live rat brain tissue for interrogation with single-cell electrophysiology and two-photon imaging techniques. Our data revealed several new observations of tissue remodeling surrounding devices: (a) there was significant disruption of dendritic arbors in neurons near implants, where losses were driven asymmetrically on the implant-facing side. (b) There was a significant loss of dendritic spine densities in neurons near implants, with a shift toward more immature (nonfunctional) morphologies. (c) There was a reduction in excitatory neurotransmission surrounding implants, as evidenced by a reduction in the frequency of excitatory postsynaptic currents (EPSCs). Lastly, (d) there were changes in the electrophysiological underpinnings of neuronal spiking regularity. In parallel, we initiated new studies to explore changes in gene expression surrounding devices through spatial transcriptomics, which we applied to both recording and stimulating arrays. We found that (a) device implantation is associated with the induction of hundreds of genes associated with neuroinflammation, glial reactivity, oligodendrocyte function, and cellular metabolism and (b) electrical stimulation induces gene expression associated with damage or plasticity in a manner dependent upon the intensity of the applied stimulus. We are currently developing computational analysis tools to distill biomarkers of device-tissue interactions from large transcriptomics data sets. These results improve the current understanding of the biological response to electrodes implanted in the brain while producing new biomarkers for benchmarking the effects of novel electrode designs on responses. As the next generation of neurotechnology is developed, it will be increasingly important to understand the influence of novel materials, surface chemistries, and implant architectures on device performance as well as the relationship with the induction of specific cellular signaling pathways.

A novel mutation in hERG gene associated with azithromycin-induced acquired long QT syndrome.

Mutations in human ether-à-go-go-related gene (hERG) potassium channels are closely associated with long QT syndrome (LQTS). Previous studies have demonstrated that macrolide antibiotics increase the risk of cardiovascular diseases. To date, the mechanisms underlying acquired LQTS remain elusive. A novel hERG mutation I1025N was identified in an azithromycin-treated patient with acquired long QT syndrome via Sanger sequencing. The mutant I1025N plasmid was transfected into HEK-293 cells, which were subsequently incubated with azithromycin. The effect of azithromycin and mutant I1025N on the hERG channel was evaluated via western blot, immunofluorescence, and electrophysiology techniques. The protein expression of the mature hERG protein was down-regulated, whereas that of the immature hERG protein was up-regulated in mutant I1025N HEK-293 cells. Azithromycin administration resulted in a negative effect on the maturation of the hERG protein. Additionally, the I1025N mutation exerted an inhibitory effect on hERG channel current. Moreover, azithromycin inhibited hERG channel current in a concentration-dependent manner. The I1025N mutation and azithromycin synergistically decreased hERG channel expression and hERG current. However, the I1025N mutation and azithromycin did not alter channel gating dynamics. These findings suggest that hERG gene mutations might be involved in the genetic susceptibility mechanism underlying acquired LQTS induced by azithromycin.

The 5HT2b Receptor in Alzheimer's Disease: Increased Levels in Patient Brains and Antagonist Attenuation of Amyloid and Tau Induced Dysfunction.

Background: Neurodegenerative diseases manifest behavioral dysfunction with disease progression. Intervention with neuropsychiatric drugs is part of most multi-drug treatment paradigms. However, only a fraction of patients responds to the treatments and those responding must deal with drug-drug interactions and tolerance issues generally attributed to off-target activities. Recent efforts have focused on the identification of underexplored targets and exploration of improved outcomes by treatment with selective molecular probes. As part of ongoing efforts to identify and validate additional targets amenable to therapeutic intervention, we examined levels of the serotonin 5-HT2b receptor (5-HT2bR) in Alzheimer's disease (AD) brains and the potential of a selective 5-HT2bR antagonist to counteract synaptic plasticity and memory damage induced by AD-related proteins, amyloid-β, and tau. This work used a combination of biochemical, chemical biology, electrophysiological, and behavioral techniques. Biochemical methods included analysis of protein levels. Chemical biology methods included the use of an in vivo molecular probe MW071, a selective antagonist for the 5HT2bR. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated spatial memory and associative memory. 5HT2bR levels are increased in brain specimens of AD patients compared to controls. 5HT2bR antagonist treatment rescued amyloid-β and tau oligomer-induced impairment of synaptic plasticity and memory. The increased levels of 5HT-2bR in AD patient brains and the attenuation of disease-related synaptic and behavioral dysfunctions by MW071 treatment suggest that the 5HT-2bR is a molecular target worth pursuing as a potential therapeutic target.

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.

Analysis of EEG characteristics in Alzheimer’s disease

Alzheimer’s disease (AD) brings a heavy burden to society and families because of the high misdiagnosis rate, prevalence rate, and disability rate. At present, the treatment of AD still faces great challenges. Early intervention can effectively delay the disease progression and improve the burden of symptom care. However, the key to early intervention is accurately identifying and assessing the disease. The examination is difficult to be widely used because of its high invasive cost. There is an urgent need for a simple, convenient, and non-invasive diagnosis of AD. Studies have shown that the brain number of AD patients is slower than that of healthy people, and the complexity of EEG is disturbed. Clinical practice has proved that the specificity and sensitivity of α, β, δ, θ absolute power and α/θ absolute power ratio, especially θ absolute power and α/θ absolute power ratio in the model, are important electrophysiological indexes for AD diagnosis EEG. Non-invasive and economical electrophysiological techniques can characterize and identify the EEG characteristics of AD patients from different angles. They are relatively simple to implement, and patients have a high degree of cooperation. Therefore, EEG, as a diagnostic method of AD, can improve the accuracy of differential diagnosis and early detection of AD and quantify the severity of the disease. It has the advantage of non-invasive convenience and price, which is of great significance for early diagnosis of AD.

Neuron-microelectrode junction induced by an engineered synapse organizer

The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1β and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording.

Neural correlates of recalled sadness, joy, and fear states: a source reconstruction EEG study.

The capacity to understand the others' emotional states, particularly if negative (e.g. sadness or fear), underpins the empathic and social brain. Patients who cannot express their emotional states experience social isolation and loneliness, exacerbating distress. We investigated the feasibility of detecting non-invasive scalp-recorded electrophysiological signals that correspond to recalled emotional states of sadness, fear, and joy for potential classification. The neural activation patterns of 20 healthy and right-handed participants were studied using an electrophysiological technique. Analyses were focused on the N400 component of Event-related potentials (ERPs) recorded during silent recall of subjective emotional states; Standardized weighted Low-resolution Electro-magnetic Tomography (swLORETA) was employed for source reconstruction. The study classified individual patterns of brain activation linked to the recollection of three distinct emotional states into seven regions of interest (ROIs). Statistical analysis (ANOVA) of the individual magnitude values revealed the existence of a common emotional circuit, as well as distinct brain areas that were specifically active during recalled sad, happy and fearful states. In particular, the right temporal and left superior frontal areas were more active for sadness, the left limbic region for fear, and the right orbitofrontal cortex for happy affective states. In conclusion, this study successfully demonstrated the feasibility of detecting scalp-recorded electrophysiological signals corresponding to internal and subjective affective states. These findings contribute to our understanding of the emotional brain, and have potential applications for future BCI classification and identification of emotional states in LIS patients who may be unable to express their emotions, thus helping to alleviate social isolation and sense of loneliness.