The evolution of patch-clamp electrophysiology: robotic, multiplex, and dynamic.

The patch-clamp technique has been the gold standard for analysis of excitable cells. Since its development in the 1980s it has contributed immensely to our understanding of neurons, muscle cells, and cardiomyocytes, and the ion channels and receptors that reside within them. This technique, predicated on Ohm's law, enables precise measurements of macroscopic excitability patterns, and ionic and gating conductances that can be assessed even down to the single channel level. Over the years, patch-clamp electrophysiology has undergone extensive modifications, with the introduction of new applications that have enhanced its power and reach. The most recent evolution of this technique occurred with the introduction of robotic high throughput automated platforms that enable high quality simultaneous recordings, in both voltage- and current-clamp modes, from 10s to 100s of cells, including cells freshly isolated from their native tissues. Combined with new dynamic-clamp applications, these new methods provide increasingly powerful tools for studying the contributions of ion channels and receptors to electrogenesis. In this brief review, we provide an overview of these enhanced patch-clamp techniques, followed by some of the applications presently being pursued, and a perspective into the potential future of the patch-clamp method. Significance Statement The patch-clamp technique, introduced in the 1980s, has revolutionized understanding of electrogenesis. Predicated on Ohm's law, this approach facilitates exploration of ionic conductances, gating mechanisms of ion channels and receptors, and their roles in neuronal, muscular, and cardiac excitability. Robotic platforms for high-throughput patch-clamp, and dynamic-clamp, have recently expanded its reach. Here, we outline new advances in patch-clamp including high throughput analysis of freshly-isolated neurons, and discuss the increasingly powerful trajectory of new patch-clamp techniques.

Excitatory action of low frequency depolarizing GABA/glycine synaptic inputs is prevalent in prenatal spinal SOD1G93A motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. Recent evidence suggests the dysfunction of inhibitory signalling in ALS motor neurons. We have shown that embryonic day (E)17.5 spinal motoneurons (MNs) of the SOD1G93A mouse model of ALS exhibit an altered chloride homeostasis. At this prenatal stage, inhibition of spinal motoneurons (MNs) is mediated by depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs). Here, using an ex vivo preparation and patch clamp recording from MNs with a chloride equilibrium set below spike threshold, we report that low input resistance (Rin ) E17.5 MNs from the SOD1G93A ALS mouse model do not correctly integrate dGPSPs evoked by electrical stimulations of GABA/glycine inputs at different frequencies. Indeed, firing activity of most wild-type (WT) MNs with low Rin was inhibited by incoming dGPSPs, whereas low Rin SOD1G93A MNs were excited or exhibited a dual response (excited by low frequency dGPSPs and inhibited by high frequency dGPSPs). Simulation highlighted the importance of the GABA/glycine input density and showed that pure excitation could be obtained in SOD-like MNs by moving GABA/glycine input away from the cell body to dendrites. This was in agreement with confocal imaging showing a lack of peri-somatic inhibitory terminals in SOD1G93A MNs compared to WT littermates. Putative fast ALS-vulnerable MNs with low Rin are therefore lacking functional inhibition at the near-term prenatal stage. KEY POINTS: We analysed the integration of GABAergic/glycinergic synaptic events by embryonic spinal motoneurons (MNs) in a mouse model of the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. We found that GABAergic/glycinergic synaptic events do not properly inhibit ALS MNs with low input resistance, most probably corresponding to future vulnerable MNs. We used a neuron model to highlight the importance of the GABA/glycine terminal location and density in the integration of the GABAergic/glycinergic synaptic events. Confocal imaging showed a lack of GABA/glycine terminals on the cell body of ALS MNs. The present study suggests that putative ALS vulnerable MNs with low Rin lack functional inhibition at the near-term stage.

Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

Nanchung and Inactive define pore properties of the native auditory transduction channel in Drosophila.

Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. Here, we established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

Role of Voltage-Gated K+ Channels and K2P Channels in Intrinsic Electrophysiological Properties and Saltatory Conduction at Nodes of Ranvier of Rat Lumbar Spinal Ventral Nerves.

Ion channels at the nodes of Ranvier (NRs) are believed to play essential roles in intrinsic electrophysiological properties and saltatory conduction of action potentials (AP) at the NRs of myelinated nerves. While we have recently shown that two-pore domain potassium (K2P) channels play a key role at the NRs of Aß-afferent nerves, K+ channels and their functions at the NRs of mammalian motor nerves remain elusive. Here we addressed this issue by using ex vivo preparations of lumbar spinal ventral nerves from both male and female rats and the pressure-patch-clamp recordings at their NRs. We found that depolarizing voltages evoked large noninactivating outward currents at NRs. The outward currents could be partially inhibited by voltage-gated K+ channel blockers, largely inhibited by K2P blockers and cooling temperatures. Inhibition of the outward currents by voltage-gated K+ channel blockers, K2P blockers, or cooling temperatures significantly altered electrophysiological properties measured at the NRs, including resting membrane potential, input resistance, AP width, AP amplitude, AP threshold, and AP rheobase. Furthermore, K2P blockers and cooling temperatures significantly reduced saltatory conduction velocity and success rates of APs in response to high-frequency stimulation. Voltage-gated K+ channel blockers reduced AP success rates at high-frequency stimulation without significantly affecting saltatory conduction velocity. Collectively, both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. The effects of cooling temperatures on saltatory conduction are at least partially mediated by K2P channels at the NRs.SIGNIFICANCE STATEMENT Ion channels localized at the NRs are believed to be key determinants of saltatory conduction on myelinated nerves. However, ion channels and their functions at the NRs have not been fully studied in different types of mammalian myelinated nerves. Here we use the pressure-patch-clamp recordings to show that both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of lumbar spinal ventral nerves of rats. Furthermore, cooling temperatures exert effects on saltatory conduction via inhibition of ion channels at the NRs. Our results provide new insights into saltatory conduction on myelinated nerves and may have physiological as well as pathologic implications.

Inhibition of suplatast tosilate on EPSC in the single paratracheal ganglion neurons attached with presynaptic boutons.

Using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with nystatin-perforated patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. EPSC frequency was higher sensitive to suplatast than EPSC amplitude. IC50 for EPSC frequency was 1.1 × 10-5 M, being similar to that for the effect on histamine release from mast cells and lower than that for the inhibitory effect on cytokine production. Suplatast also inhibited the EPSCs potentiated by bradykinin (BK), but it did not affect the potentiation itself by BK. Thus suplatast inhibited the EPSC of PTG neurons attached with presynaptic boutons at both the presynaptic and postsynaptic sites.NEW & NOTEWORTHY In this study, using single neurons of rat paratracheal ganglia (PTG) attached with presynaptic boutons, the effects of suplatast tosilate on excitatory postsynaptic currents (EPSCs) were investigated with patch-clamp recording technique. We found that suplatast concentration dependently inhibited the EPSC amplitude and its frequency in single PTG neurons attached with presynaptic boutons. Thus suplatast inhibited the function of PTG neurons at both of presynaptic and postsynaptic sites.

Activin A targets extrasynaptic NMDA receptors to ameliorate neuronal and behavioral deficits in a mouse model of Huntington disease.

Cortical-striatal synaptic dysfunction, including enhanced toxic signaling by extrasynaptic N-methyl-d-aspartate receptors (eNMDARs), precedes neurodegeneration in Huntington disease (HD). A previous study showed Activin A, whose transcription is upregulated by calcium influx via synaptic NMDARs, suppresses eNMDAR signaling. Therefore, we examined the role of Activin A in the YAC128 HD mouse model, comparing it to wild-type controls. We found decreased Activin A secretion in YAC128 cortical-striatal co-cultures, while Activin A overexpression in this model rescued altered eNMDAR expression. Striatal overexpression of Activin A in vivo improved motor learning on the rotarod task, and normalized striatal neuronal eNMDAR-mediated currents, membrane capacitance and spontaneous excitatory postsynaptic current frequency in the YAC128 mice. These results support the therapeutic potential of Activin A signaling and targeting eNMDARs to restore striatal neuronal health and ameliorate behavioral deficits in HD.

Developing a Novel Method for the Analysis of Spinal Cord-Penile Neurotransmission Mechanisms.

Sexual dysfunction can be caused by impaired neurotransmission from the peripheral to the central nervous system. Therefore, it is important to evaluate the input of sensory information from the peripheral genital area and investigate the control mechanisms in the spinal cord to clarify the pathological basis of sensory abnormalities in the genital area. However, an in vivo evaluation system for the spinal cord-penile neurotransmission mechanism has not yet been developed. Here, urethane-anesthetized rats were used to evaluate neuronal firing induced by innocuous or nociceptive stimulation of the penis using extracellular recording or patch-clamp techniques in the lumbosacral spinal dorsal horn and electrophysiological evaluation in the peripheral pelvic nerves. As a result, innocuous and nociceptive stimuli-evoked neuronal firing was successfully recorded in the deep and superficial spinal dorsal horns, respectively. The innocuous stimuli-evoked nerve firing was also recorded in the pelvic nerve. These firings were suppressed by lidocaine. To the best of our knowledge, this is the first report of a successful quantitative evaluation of penile stimuli-evoked neuronal firing. This method is not only useful for analyzing the pathological basis of spinal cord-penile neurotransmission in sexual dysfunction but also provides a useful evaluation system in the search for new treatments.

Biocytin-Labeling in Whole-Cell Recording: Electrophysiological and Morphological Properties of Pyramidal Neurons in CYLD-Deficient Mice.

Biocytin, a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine, has been used as a histological dye to stain nerve cells. Electrophysiological activity and morphology are two key characteristics of neurons, but revealing both the electrophysiological and morphological properties of the same neuron is challenging. This article introduces a detailed and easy-to-operate procedure for single-cell labeling in combination with whole-cell patch-clamp recording. Using a recording electrode filled with a biocytin-containing internal solution, we demonstrate the electrophysiological and morphological characteristics of pyramidal (PNs), medial spiny (MSNs) and parvalbumin neurons (PVs) in brain slices, where the electrophysiological and morphological properties of the same individual cell are elucidated. We first introduce a protocol for whole-cell patch-clamp recording in various neurons, coupled with the intracellular diffusion of biocytin delivered by the glass capillary of the recording electrode, followed by a post hoc procedure to reveal the architecture and morphology of biocytin-labeled neurons. An analysis of action potentials (APs) and neuronal morphology, including the dendritic length, number of intersections, and spine density of biocytin-labeled neurons, were performed using ClampFit and Fiji Image (ImageJ), respectively. Next, to take advantage of the techniques introduced above, we uncovered defects in the APs and the dendritic spines of PNs in the primary motor cortex (M1) of deubiquitinase cylindromatosis (CYLD) knock-out (Cyld-/-) mice. In summary, this article provides a detailed methodology for revealing the morphology as well as the electrophysiological activity of a single neuron that will have many applications in neurobiology.

Changes of the biophysical properties of voltage-gated Na+ currents during maturation of the sodium-taste cells in rat fungiform papillae.

Taste cells are a heterogeneous population of sensory receptors that undergo continuous turnover. Different chemo-sensitive cell lines rely on action potentials to release the neurotransmitter onto nerve endings. The electrical excitability is due to the presence of a tetrodotoxin-sensitive, voltage-gated sodium current (INa ) similar to that found in neurons. Since the biophysical properties of neuronal INa change during development, we wondered whether the same also occurred in taste cells. Here, we used the patch-clamp recording technique to study INa in salt-sensing cells (sodium cells) of rat fungiform papillae. We identified these cells by exploiting the known blocking effect of amiloride on ENaC, the sodium (salt) receptor. Based on the amplitude of INa , which is known to increase during development, we subdivided sodium cells into two groups: cells with small sodium current (SSC cells; INa  < 1 nA) and cells with large sodium current (LSC cells; INa  > 1 nA). We found that: the voltage dependence of activation and inactivation significantly differed between these subsets; a slowly inactivating sodium current was more prominent in LSC cells; membrane capacitance in SSC cells was larger than in LSC cells. mRNA expression analysis of the α-subunits of voltage-gated sodium channels in fungiform taste buds supported the functional data. Lucifer Yellow labelling of recorded cells revealed that our electrophysiological criterion for distinguishing two broad groups of taste cells was in good agreement with morphological observations for cell maturity. Thus, all these findings are consistent with developmental changes in the voltage-dependent properties of sodium-taste cells. KEY POINTS: Taste cells are sensory receptors that undergo continuous turnover while they detect food chemicals and communicate with afferent nerve fibres. The voltage-gated sodium current (INa ) is a key ion current for generating action potentials in fully differentiated and chemo-sensitive taste cells, which use electrical signalling to release neurotransmitters. Here we show that, during the maturation of rat taste cells involved in salt detection (sodium cells), the biophysical properties of INa , such as voltage dependence of activation and inactivation, change significantly. Our results help reveal how taste cells gain electrical excitability during turnover, a property critical to their operation as chemical detectors that relay sensory information to nerve fibres.

Tyrosine 288 in the extracellular domain of the human P2X7 receptor is critical for receptor function revealed by structural modeling and site-directed mutagenesis.

The P2X7 receptor (P2X7R) is a calcium-permeable cation channel activated by high concentrations of extracellular ATP. It plays a role in vital physiological processes, particularly in innate immunity, and is dysregulated in pathological conditions such as inflammatory diseases, neurodegenerative diseases, mood disorders, and cancers. Structural modeling of the human P2X7R (hP2X7R) based on the recently available structures of the rat P2X7 receptor (rP2XR) in conjunction with molecular docking predicts the orientation of tyrosine at position 288 (Y288) in the extracellular domain to face ATP. In this short communication, we combined site-directed mutagenesis and whole-cell patch-clamp recording to investigate the role of this residue in the hP2X7R function. Mutation of this extracellular residue to amino acids with different properties massively impaired current responses to both ATP and BzATP, suggesting that Y288 is important for normal receptor function. Such a finding facilitates development of an in-depth understanding of the molecular basis of hP2X7R structure-function relationships.

Complete Freund's adjuvant-induced protein dysregulation correlated with mirror image pain as assessed by quantitative proteomics of the mouse spinal cord.

Inflammation or trauma occurring on one side of the body can cause pathological pain on the contralateral noninjured side in a phenomenon called mirror-image pain (MIP). Although some potential mechanisms involved in MIP have been reported, including those involving the immune system and glial cells as well as neural mechanisms, the molecular mechanisms are not well understood. In this study, we aimed to understand the molecular mechanisms in MIP using quantitative proteomics and whole-cell patch clamp recordings. Behavioral test results showed that complete Freund's adjuvant could induce MIP in the mice. The results of isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomics showed that 108 proteins were dysregulated, and these proteins may represent potential targets. Furthermore, bioinformatics analysis was applied to explore the potential molecular mechanisms during MIP after complete Freund's adjuvant (CFA) treatment. Parallel reaction monitoring (PRM) results showed that PKCδ and seven other dysregulated proteins were related to MIP after CFA treatment. Patch clamp recording results showed that CFA treatment could increase intrinsic excitability and spontaneous firing in spinal cord neurons during MIP. In summary, we found that CFA could induce MIP. The results of proteomic research on the spinal cord after CFA treatment could provide new insight into the molecular mechanisms of MIP. Moreover, the neuronal activity of spinal cord neurons was upregulated during MIP after CFA treatment. In summary, the results of the spinal cord proteomic profile provide a potential molecular mechanism for understanding MIP.

Adenosine A1 receptor antagonist-induced facilitation of postsynaptic AMPA currents in pyramidal neurons of the rat hippocampal CA2 area.

Adenosine A1 receptors (A1R) are widely expressed in hippocampal pyramidal neurons and their presynaptic terminals. It is well known that endogenous adenosine regulates hippocampal function through the activation of A1R in hippocampal pyramidal neurons and has been reported that blockade of A1R induces stronger potentiation of excitatory synaptic transmission in CA2 pyramidal neurons than in CA1 pyramidal neurons. This strong potentiation of CA2 neurons is thought to be caused by the specific modulation of excitatory synaptic transmission through postsynaptic A1R. However, the direct effects of A1R on postsynaptic AMPA channels remain unknown because of the technical difficulties of patch-clamp recording from mature hippocampal CA2 neurons. We recorded synaptic currents from pyramidal neurons in CA1 and CA2 and analyzed the effects of an A1R antagonist on stimulation-evoked synaptic transmission and local application-induced postsynaptic AMPA currents. The antagonist increased the amplitude of evoked synaptic transmission in neurons in both CA1 and CA2. This facilitation was larger in pyramidal neurons in CA2 than in CA1. The antagonist also increased postsynaptic AMPA currents in neurons in CA2 but not in CA1. This facilitation of CA2 AMPA currents was occluded by the intracellular application of a G-protein blocker. Even with the blockade of postsynaptic G-protein signaling, the A1R antagonist increased evoked synaptic transmission in neurons in CA2. These results suggest that synaptic transmission in pyramidal neurons in CA2 is regulated by both presynaptic and postsynaptic A1R. Moreover, A1R regulate excitatory synaptic transmission in pyramidal neurons in CA2 through the characteristic postsynaptic modulation of AMPA currents.

Distinct effects of orexin A on spontaneous and evoked synaptic currents in the rat nucleus tractus solitarius.

Orexins are produced in hypothalamic areas and orexin-containing neurons are distributed in widespread areas of the central nervous system. Orexins regulate several physiological functions such as arousal, food intake and autonomic control. The presence of orexin-containing neuron terminals and orexin receptors has been confirmed in the nucleus tractus solitarius (NTS), which receives primary afferent fibers from peripheral organs including baroreceptors. However, the neuronal effects of orexin-1 receptor (OX1R) activation were not examined. Here, we aimed to determine the effects of OX1R activation on excitatory synaptic transmission. OX1R activation increased the frequency of spontaneous excitatory synaptic currents (sEPSCs). This effect was blocked by the prior application of L-NAME. In contrast, the amplitude of evoked excitatory postsynaptic currents (eEPSCs) was suppressed by OX1R activation, and this effect was prevented by a cannabinoid receptor 1 blocker, AM251, but not by the pretreatment with L-NAME. Altogether, these results suggest that OX1R activation increases sEPSCs frequency by stimulating NO production, whereas it inhibits eEPSCs by releasing endocannabinoids in the NTS. Thus, OX1R activation had distinct effects on spontaneous and evoked excitatory synaptic transmissions in the NTS.

Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

Two-dimensional Ti3C2Tx MXene promotes electrophysiological maturation of neural circuits.

BACKGROUND: The ideal neural interface or scaffold for stem cell therapy shall have good biocompatibility promoting survival, maturation and integration of neural stem cells (NSCs) in targeted brain regions. The unique electrical, hydrophilic and surface-modifiable properties of Ti3C2Tx MXene make it an attractive substrate, but little is known about how it interacts with NSCs during development and maturation. RESULTS: In this study, we cultured NSCs on Ti3C2Tx MXene and examined its effects on morphological and electrophysiological properties of NSC-derived neurons. With a combination of immunostaining and patch-clamp recording, we found that Ti3C2Tx MXene promotes NSCs differentiation and neurite growth, increases voltage-gated current of Ca2+ but not Na+ or K+ in matured neurons, boosts their spiking without changing their passive membrane properties, and enhances synaptic transmission between them. CONCLUSIONS: These results expand our understanding of interaction between Ti3C2Tx MXene and NSCs and provide a critical line of evidence for using Ti3C2Tx MXene in neural interface or scaffold in stem cell therapy.

Dopamine Signaling in Wake-Promoting Clock Neurons Is Not Required for the Normal Regulation of Sleep in Drosophila.

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNvs) and small ventrolateral neurons (s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D1-like dopamine receptor (Dop1R1) in the l-LNvs and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Downregulation of Dop1R1 or Dop1R2 in the l-LNvs and s-LNvs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNvs do not use Dop1R2 receptors and the s-LNvs also respond to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs.SIGNIFICANCE STATEMENT In insect and mammalian brains, sleep-promoting networks are intimately linked to the circadian clock, and the mechanisms underlying sleep and circadian timekeeping are evolutionarily ancient and highly conserved. Here we show that dopamine, one important sleep modulator in flies and mammals, plays surprisingly complex roles in the regulation of sleep by clock-containing neurons. Dopamine inhibits neurons in a central brain sleep center to promote sleep and excites wake-promoting circadian clock neurons. It is therefore predicted to promote wakefulness through both of these networks. Nevertheless, our results reveal that dopamine acting on wake-promoting clock neurons promotes sleep, revealing a previously unappreciated complexity in the dopaminergic control of sleep.

Downstream projection of Barrington's nucleus to the spinal cord in mice.

Barrington's nucleus (Bar), which controls micturition behavior through downstream projections to the spinal cord, contains two types of projection neurons, BarCRH and BarESR1, that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus, whereas BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory postsynaptic currents (oEPSCs) in 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiI)-labeled lumbosacral preganglionic neurons (LS-PGNs) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGNs, whereas BarESR1 neurons also elicited smaller-amplitude glutamatergic polysynaptic oEPSCs or polysynaptic opto-evoked inhibitory postsynaptic currents (oIPSCs) in some LS-PGNs. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting to either the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous postsynaptic currents in LS-PGNs. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.NEW & NOTEWORTHY Photostimulation of BarCRH or BarESR1 axons in the adult mouse spinal cord elicits excitatory or inhibitory postsynaptic responses in multiple cell types related to the autonomic nervous system including preganglionic neurons (PGNs) in the lumbosacral intermediolateral nucleus and interneurons in the lumbosacral dorsal commissure nucleus. Integration of excitatory inputs from Bar and from visceral primary afferents in PGNs may be important in the regulation of micturition behavior.

Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABAA receptors (GABAA Rs). In this study, the modulatory potential of 11 SQTs at GABAA Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of ß-caryophyllene and α-humulene, as well as their respective derivatives ß-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABAA R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ2 and δ subunits is important for SQT modulation. While phasic GABAA receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α1 ß2 γ2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABAA R localized between transmembrane segments 1 and 3 at the (+ α)-(- α) interface. In sum, differences in the modulation of GABAA R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds.

In Vivo Whole-Cell Patch-Clamp Methods: Recent Technical Progress and Future Perspectives.

Brain functions are fundamental for the survival of organisms, and they are supported by neural circuits consisting of a variety of neurons. To investigate the function of neurons at the single-cell level, researchers often use whole-cell patch-clamp recording techniques. These techniques enable us to record membrane potentials (including action potentials) of individual neurons of not only anesthetized but also actively behaving animals. This whole-cell recording method enables us to reveal how neuronal activities support brain function at the single-cell level. In this review, we introduce previous studies using in vivo patch-clamp recording techniques and recent findings primarily regarding neuronal activities in the hippocampus for behavioral function. We further discuss how we can bridge the gap between electrophysiology and biochemistry.