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Vincristine-induced peripheral neuropathy is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine-induced pain. Our previous studies have shown that the tetrodotoxin-sensitive voltage-gated sodium channel Nav1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) Nav1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons, we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in the half-maximal potential (V1/2) of activation of Nav1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from Nav1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel Nav1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Electric fields affect the activity of neurons and brain circuits, yet how this happens at the cellular level remains enigmatic. Lack of understanding of how to stimulate the brain to promote or suppress specific activity significantly limits basic research and clinical applications. Here, we study how electric fields impact subthreshold and spiking properties of major cortical neuronal classes. We find that neurons in the rodent and human cortex exhibit strong, cell-class-dependent entrainment that depends on stimulation frequency. Excitatory pyramidal neurons, with their slower spike rate, entrain to both slow and fast electric fields, while inhibitory classes like Pvalb and Sst (with their fast spiking) predominantly phase-lock to fast fields. We show that this spike-field entrainment is the result of two effects: non-specific membrane polarization occurring across classes and class-specific excitability properties. Importantly, these properties are present across cortical areas and species. These findings allow for the design of selective and class-specific neuromodulation., Competing Interests: Declaration of interests S.Y.L. has previously consulted for Starfish Neuroscience, Inc. C.A.A. and S.Y.L. are listed as inventors on a patent application related to this work. C.K. is a board member of and has a financial interest in Intrinsic Powers, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel Na V 1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of Na V 1.6 G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant Na V 1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of Na V 1.6 G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in Na V 1.6 G1625R . Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Variants in SCN8A are associated with a spectrum of epilepsies and neurodevelopmental disorders. Ataxia as a predominant symptom of SCN8A variation has not been well studied. We set out to investigate disease mechanisms and genotype-phenotype correlations of SCN8A-related ataxia., Methods: We collected genetic and electro-clinical data of ten individuals from nine unrelated families carrying novel SCN8A variants associated with chronic progressive or episodic ataxia. Electrophysiological characterizations of these variants were performed in ND7/23 cells and cultured neurons., Findings: Variants associated with chronic progressive ataxia either decreased Na + current densities and shifted activation curves towards more depolarized potentials (p.Asn995Asp, p.Lys1498Glu and p.Trp1266Cys) or resulted in a premature stop codon (p.Trp937Ter). Three variants (p.Arg847Gln and biallelic p.Arg191Trp/p.Asp1525Tyr) were associated with episodic ataxia causing loss-of-function by decreasing Na + current densities or a hyperpolarizing shift of the inactivation curve. Two additional episodic ataxia-associated variants caused mixed gain- and loss-of function effects in ND7/23 cells and were further examined in primary murine hippocampal neuronal cultures. Neuronal firing in excitatory neurons was increased by p.Arg1629His, but decreased by p.Glu1201Lys. Neuronal firing in inhibitory neurons was decreased for both variants. No functional effect was observed for p.Arg1913Trp. In four individuals, treatment with sodium channel blockers exacerbated symptoms., Interpretation: We identified episodic or chronic ataxia as predominant phenotypes caused by variants in SCN8A. Genotype-phenotype correlations revealed a more pronounced loss-of-function effect for variants causing chronic ataxia. Sodium channel blockers should be avoided under these conditions., Funding: BMBF, DFG, the Italian Ministry of Health, University of Tuebingen., Competing Interests: Declaration of interests MK reports a doctoral grant from DAAD. JDOE is coordinator of the Chorea and Huntington Disease Group (ERN-RND). EB has received funding from the Dutch Epilepsy Foundation, on behalf of the Dravet syndrome Foundation Netherlands/Flanders, the JANIVO foundation and the K.F. Hein Foundation. LS declares grants from the German Research Foundation, German Ministry of Heath, European Commission, and Innovationsfond, and has received consulting fees from Vico Therapeutics and Novartis. RSM has received consulting and speaker fees from UCB and Orion and speaker fees from EISAI and Angelini Pharma. All other authors report no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

During the critical period of postnatal development, brain maturation is extremely sensitive to external stimuli. Newborn rodents already have functional somatosensory pathways and the thalamus, but the cortex is still forming. Immature thalamic synapses may produce large postsynaptic potentials in immature neurons, while non-synaptic membrane currents remain relatively weak and slow. The thalamocortical system generates spontaneous and evoked early gamma and spindle-burst oscillations in newborn rodents. How relatively strong synapses and weak intrinsic currents interact with each other and how they contribute to early thalamic activities remains largely unknown. Here, we performed local field potential (LFP), juxtacellular, and patch-clamp recordings in the somatosensory thalamus of urethane-anesthetized rat pups at postnatal days 6-7 with one whisker stimulation. We removed the overlying cortex and hippocampus to reach the thalamus with electrodes. Deflection of only one (the principal) whisker induced spikes in a particular thalamic cell. Whisker deflection evoked a group of large-amplitude excitatory events, likely originating from lemniscal synapses and multiple inhibitory postsynaptic events in thalamocortical cells. Large-amplitude excitatory events produced a group of spike bursts and could evoke a depolarization block. Juxtacellular recordings confirmed the partial inactivation of spikes. Inhibitory events prevented inactivation of action potentials and gamma-modulated neuronal firing. We conclude that the interplay of strong excitatory and inhibitory synapses and relatively weak intrinsic currents produces sensory-evoked early gamma oscillations in thalamocortical cells. We also propose that sensory-evoked large-amplitude excitatory events contribute to evoked spindle-bursts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

The patch-clamp technique is the gold-standard methodology for analysis of excitable cells. However, throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for applications like drug screening, has been primarily used to study channels and receptors expressed in heterologous systems. We introduce an approach for automated high-throughput patch-clamping that enhances analysis of excitable cells at the channel and cellular levels. This involves dissociating and isolating neurons from intact tissues and patch-clamping using a robotic instrument, followed by using an open-source Python script for analysis and filtration. As a proof of concept, we apply this approach to investigate the biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables voltage- and current-clamp recordings in the same cell, allowing unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of freshly isolated neurons without requiring culturing on coverslips., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

The mechanisms underlying the antiarrhythmic action of compound trihydrochloride N 1 -(2,3,4-trimethoxy)-N 2 -{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) were studied in vitro. The experiments were performed on a culture of rat hippocampal neurons. The electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It is shown that the compound ALM-802 effectively blocks potential-dependent Na + and K + channels and does not affect the activity of potential-dependent Ca 2+ channels. The inhibition of currents through these channels is dose-dependent; the IC 50 of Na + and K + channels were 94±4 and 67±3 μM, respectively. These findings indicate that compound ALM-802 combines the properties of class I and class III antiarrhythmic agents according to the Vaughan-Williams classification., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

The functioning of voltage-dependent K channels (Kv) may correlate with the physiological state of brain in organisms, including the sleep in Drosophila. Apparently, all major types of K currents are expressed in CNS of this model organism. These are the Shab-Kv2, Shaker-Kv1, Shal-Kv4, and Shaw-Kv3 α subunits and can be deciphered by patch-clamp technique. Although it is plausible that some of these channels may play a prevailing role in sleep or wakefulness, several of recent data are not conclusive. It needs to be defined that indeed the frequency of action potentials in large ventral lateral pacemaker neurons is either higher or lower during the morning or night because of an increased Kv3 and Kv4 currents, respectively. The outcomes of dynamic-clamp approach in combination with electrophysiology in insects are unreliable in contrast to those in mammalian neurons. Since the addition of virtual Kv conductance during any Zeitgeber time should not significantly alter the resting membrane potential. This review explains the Drosophila sleep behavior based on neural activity with respect to K current-driven action potential rate., (© 2022 Wiley Periodicals LLC.)

In in vitro experiments on isolated rat hippocampal neurons, we studied the electrophysiological mechanisms of the antiarrhythmic effects of N-deacetyllappaconitine monochlorhydrate (DALCh), active metabolite of lappaconitine hydrobromide (allapinin). Electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It was shown that DALCh increased the duration of both slow and fast depolarization phases and decreased the amplitude of the action potential. DALCh effectively inhibited transmembrane currents of Na + ions and partially K + ions through the corresponding transmembrane voltage-gated ion channels. Thus, DALCh, in contrast to lappaconitine hydrobromide, belongs not to 1C, but to the 1A class of antiarrhythmics according to the Vaughan-Williams classification., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Long-term manipulation of activity in the neonatal rodent brain can help us understand healthy development, but also involves a set of challenges unique to the neonatal animal. As pups are small, cannot be separated from their mother for long periods of time, and must be housed in a nest, many traditional techniques are unusable during the first two postnatal weeks., New Method: Here, we describe the use of magnetic resonance induction to allow wireless and chronic optogenetic manipulation of spontaneous activity in mouse pups during the second postnatal week., Results: Pups were implanted with a lightweight receiver coupled to an LED and successfully returned to the homecage. A transmitter coil surrounding the homecage drove the implanted LED and was regulated by a microcontroller to allow flexible, precisely-timed and wireless control over neuronal manipulation. In vivo patch-clamp recordings verified that activation of the LED triggered bursts of action potentials in layer 2/3 neurons that expressed channelrhodopsin-2 in the visual cortex without directly affecting neighboring, non-expressing neurons. The implants are stable and functional for at least 10 days and do not have an impact on the weight gain of pups. Implanted pups' behavior is mildly affected only briefly after surgery, while maternal behavior of dams remains unaffected., Comparison With Existing Method(s): In contrast to most other methods for wireless optogenetic stimulation, the small size and low weight of the receiver allow complete implantation in animals that are as small as a newborn mouse., Conclusions: This method is ideal for investigating the function and development of cortical circuits in small and developing animals. Furthermore, our method is economical and easy to adapt to diverse experimental designs., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5-15 min after formalin injection) but not during the later phase (phase 2, 15-60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0-5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In temporal lobe epilepsy, the ability of the dentate gyrus to limit excitatory cortical input to the hippocampus breaks down, leading to seizures. The dentate gyrus is also thought to help discriminate between similar memories by performing pattern separation, but whether epilepsy leads to a breakdown in this neural computation, and thus to mnemonic discrimination impairments, remains unknown. Here we show that temporal lobe epilepsy is characterized by behavioral deficits in mnemonic discrimination tasks, in both humans (females and males) and mice (C57Bl6 males, systemic low-dose kainate model). Using a recently developed assay in brain slices of the same epileptic mice, we reveal a decreased ability of the dentate gyrus to perform certain forms of pattern separation. This is because of a subset of granule cells with abnormal bursting that can develop independently of early EEG abnormalities. Overall, our results linking physiology, computation, and cognition in the same mice advance our understanding of episodic memory mechanisms and their dysfunction in epilepsy. SIGNIFICANCE STATEMENT People with temporal lobe epilepsy (TLE) often have learning and memory impairments, sometimes occurring earlier than the first seizure, but those symptoms and their biological underpinnings are poorly understood. We focused on the dentate gyrus, a brain region that is critical to avoid confusion between similar memories and is anatomically disorganized in TLE. We show that both humans and mice with TLE experience confusion between similar situations. This impairment coincides with a failure of the dentate gyrus to disambiguate similar input signals because of pathologic bursting in a subset of neurons. Our work bridges seizure-oriented and memory-oriented views of the dentate gyrus function, suggests a mechanism for cognitive symptoms in TLE, and supports a long-standing hypothesis of episodic memory theories., (Copyright © 2021 the authors.)

Epileptic activity leads to rapid insertion of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) into the synapses of cortical and hippocampal glutamatergic neurons, which generally do not express them. The physiological significance of this process is not yet fully understood; however, it is usually assumed to be a pathological process that augments epileptic activity. Using whole-cell patch-clamp recordings in rat entorhinal cortex slices, we demonstrate that the timing of epileptiform discharges, induced by 4-aminopyridine and gabazine, is determined by the shunting effect of Ca 2+ -dependent slow conductance, mediated predominantly by K + -channels. The blockade of CP-AMPARs by IEM-1460 eliminates this extra conductance and consequently increases the rate of discharge generation. The blockade of NMDARs reduced the additional conductance to a lesser extent than the blockade of CP-AMPARs, indicating that CP-AMPARs are a more significant source of intracellular Ca 2+ . The study's main findings were implemented in a mathematical model, which reproduces the shunting effect of activity-dependent conductance on the generation of discharges. The obtained results suggest that the expression of CP-AMPARs in principal neurons reduces the discharge generation rate and may be considered as a protective mechanism.

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca 2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic β-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca 2+ entry to larger or lesser extent. Potency follows the order 3 R, 4 R, 2' R > 3 S, 4 S, 2' R ≅ 3 R, 4 R, 2' S > 3 S, 4 S, 2' S, with the most potent diastereoisomer showing a half inhibitory concentration (IC 50 ) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these β-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N -(3-aminopropyl)-2-[(3-methylphenyl)methoxy]- N -(2-thienylmethyl)-benzamide (AMTB) antagonist.

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein's function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

Single-cell transcriptomic approaches are revolutionizing neuroscience. Integrating this wealth of data with morphology and physiology, for the comprehensive study of neuronal biology, requires multiplexing gene expression data with complementary techniques. To meet this need, multiple groups in parallel have developed "Patch-seq," a modification of whole-cell patch-clamp protocols that enables mRNA sequencing of cell contents after electrophysiological recordings from individual neurons and morphologic reconstruction of the same cells. In this review, we first outline the critical technical developments that enabled robust Patch-seq experimental efforts and analytical solutions to interpret the rich multimodal data generated. We then review recent applications of Patch-seq that address novel and long-standing questions in neuroscience. These include the following: (1) targeted study of specific neuronal populations based on their anatomic location, functional properties, lineage, or a combination of these factors; (2) the compilation and integration of multimodal cell type atlases; and (3) the investigation of the molecular basis of morphologic and functional diversity. Finally, we highlight potential opportunities for further technical development and lines of research that may benefit from implementing the Patch-seq technique. As a multimodal approach at the intersection of molecular neurobiology and physiology, Patch-seq is uniquely positioned to directly link gene expression to brain function., (Copyright © 2021 the authors.)

GABA A receptors (GABA A Rs) and glycine receptors (GlyRs) are two principal inhibitory chloride ion channels in the central nervous system. The two receptors do not function independently but cross-talk to each other, i.e., the activation of one receptor would inhibit the other. This cross-talk is present in different patterns across various regions in the central nervous system; however, the factor that determines these patterns is not understood. Here, we show that the pattern of cross-talk between the two receptors is shaped by their relative expression level in a neuron: a higher expression level correlates with louder talk. In line with a tendency of decrease in expression level of GlyRs and increase in expression level of GABA A Rs from the spinal cord, the brainstem to the neocortex, GlyRs talked much louder (i.e. produced greater inhibition) than GABA A Rs (one-way pattern) in spinal cord neurons, about equally loud as GABA A Rs (symmetric pattern) in inferior colliculus neurons and less loud (i.e. less inhibition) than GABA A Rs (asymmetric pattern) in auditory cortex neurons. Overexpression of GlyRs in inferior colliculus neurons produced an asymmetric pattern that should otherwise have been observed in spinal cord neurons. These expression level-dependent patterns of cross-talk between the two receptors may suggest how the central nervous system uses an alternative mechanism to maintain a delicate level of inhibition through adjusting the proportion of the two receptors in a neuron along its pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, is mainly expressed in nociceptive primary sensory neurons. Sensitivity of TRPV1 to several stimuli is known to vary among species, specifically, the avian orthologue is nearly insensitive to capsaicin. Extracellular sodium ions ([Na + ] o ) regulate TRPV1 activity in mammals, but their regulatory role on chicken TRPV1 (cTRPV1) is unknown. Here, we focused on the actions of capsaicin and low [Na + ] o on cTRPV1 activity. In chicken dorsal root ganglion (cDRG) neurons, capsaicin elicited [Ca 2+ ] i increases, but its effective concentration was much higher than those in mammals. Low [Na + ] o evoked [Ca 2+ ] i increases in cDRG neurons in a decreasing [Na + ] o -dependent manner and the complete removal of [Na + ] o (0Na) produced maximal effects. The population of 0Na-sensitive neurons was mostly overlapped with those of proton- and capsaicin-sensitive ones. Low [Na + ] o synergistically potentiated the capsaicin- and proton-induced TRPV1 activation in cDRG neurons. In HEK293 cells expressing cTRPV1 (cTRPV1-HEK), capsaicin elicited [Ca 2+ ] i increases with an EC 50 of 11.8 µM, and low [Na + ] o also did. Well-defined mammalian TRPV1 antagonists hardly suppressed cTRPV1 activation by low [Na + ] o . 0Na evoked outwardly rectified currents in cTRPV1-HEK. Mutagenesis analyses revealed a possible interaction of [Na + ] o with the proton-binding sites of cTRPV1. The administration of capsaicin and 0Na to chick eyes elicited pain-related behaviors. These results suggest that low [Na + ] o is capable of activating cTRPV1 in vitro, resulting in pain in vivo. Our data demonstrate that characterization of the cTRPV1 function is important to understand activation mechanisms of TRPV1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A rare mutation affecting the Forkhead-box protein P2 (FOXP2) transcription factor causes a severe monogenic speech and language disorder. Mice carrying an identical point mutation to that observed in affected patients (Foxp2 +/R552H mice) display motor deficits and impaired synaptic plasticity in the striatum. However, the consequences of the mutation on neuronal function, in particular in the cerebral cortex, remain little studied. Foxp2 is expressed in a subset of Layer VI cortical neurons. Here, we used Ntsr1-EGFP mice to identify Foxp2+ neurons in the mouse auditory cortex ex vivo. We studied the functional impact of the R552H mutation on the morphologic and functional properties of Layer VI cortical neurons from Ntsr1-EGFP; Foxp2 +/R552H male and female mice. The complexity of apical, but not basal dendrites was significantly lower in Foxp2 +/R552H cortico-thalamic neurons than in control Foxp2 +/+ neurons. Excitatory synaptic inputs, but not inhibitory synaptic inputs, were decreased in Foxp2 +/R552H mice. In response, homeostatic mechanisms would be expected to increase neuronal gain, i.e., the conversion of a synaptic input into a firing output. However, the intrinsic excitability of Foxp2+ cortical neurons was lower in Foxp2 +/R552H neurons. A-type and delayed-rectifier (DR) potassium currents, two putative transcriptional targets of Foxp2, were not affected by the mutation. In contrast, GABA B /GIRK signaling, another presumed target of Foxp2, was increased in mutant neurons. Blocking GIRK channels strongly attenuated the difference in intrinsic excitability between wild-type (WT) and Foxp2 +/R552H neurons. Our results reveal a novel role for Foxp2 in the control of neuronal input/output homeostasis. SIGNIFICANCE STATEMENT Mutations of the Forkhead-box protein 2 (FOXP2) gene in humans are the first known monogenic cause of a speech and language disorder. The Foxp2 mutation may directly affect neuronal development and function in neocortex, where Foxp2 is expressed. Brain imaging studies in patients with a heterozygous mutation in FOXP2 showed abnormalities in cortical language-related regions relative to the unaffected members of the same family. However, the role of Foxp2 in neocortical neurons is poorly understood. Using mice with a Foxp2 mutation equivalent to that found in patients, we studied functional modifications in auditory cortex neurons ex vivo We found that mutant neurons exhibit alterations of synaptic input and GABAB/GIRK signaling, reflecting a loss of neuronal homeostasis., (Copyright © 2020 the authors.)

As an imidazoline I1 receptor agonist with very weak binding affinity for α 2 -adrenoceptors, moxonidine is commonly used in the treatment of hypertension. Moxonidine also has been implicated to act centrally to reduce airway vagal outflow. However, it is unknown at which central sites moxonidine acts to affect airway vagal activity, and how moxonidine takes effect at synaptic and receptor levels. In this study, airway vagal preganglionic neurons (AVPNs) were retrogradely labeled in neonatal rats from the intrathoracic trachea; retrogradely labeled AVPNs in the external formation of the nucleus ambiguus (NA) were identified in rhythmically active medullary slices using whole-cell patch-clamp techniques; and the effects of moxonidine on the spontaneous excitatory postsynaptic currents (EPSCs) of AVPNs were observed at synaptic level. The results show that moxonidine (10 μmol·L -1 ) significantly inhibited the frequency of spontaneous EPSCs in both inspiratory-activated and inspiratory-inhibited AVPNs. This effect was partially blocked by SKF-86466 (10 μmol·L -1 ), a highly selective antagonist of α 2 -adrenoceptors, or AGN-192403, a selective antagonist of imidazoline I1 receptors, and was completely blocked by efaroxan (10 μmol·L -1 ), an antagonist of both α 2 -adrenoceptors and imidazoline I1 receptors. These results demonstrate that moxonidine inhibits the excitatory inputs to AVPNs via activation of both α 2 -adrenoceptors and imidazoline I1 receptors, and suggest that physiologically both of these two types of receptors are involved in the central regulation of airway vagal activity at preganglionic level. Moxonidine might be potentially useful in diseases with aberrant airway vagal activity such as asthma and chronic obstructive diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α 3 β 4 -nAChR expressed in native SH-SY5Y cells. α 3 β 4 -nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC 50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC 50 of 1.5 μM. Kinetic analysis showed that cocaine accelerated α 3 β 4 -nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 μM) depressed the maximum response on the nicotine concentration-response curve without changing the EC 50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-βS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 μM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α 6 /α 3 β 2 β 3 -nAChRs and α 4 β 2 -nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α 3 β 4 -nAChRs expressed in SH-SY5Y cells.

Peptides constituted of backbone homologated α-amino acids combined with carbon materials offer interesting possibilities in the modulation of cellular functions. In this work, we have prepared diphenylalanine β- and γ-peptides and conjugated them to carbon nanotubes (CNTs). These hybrids were able to self-assemble into fibrillar dendritic structures enabling the growth of primary hippocampal cells and the modulation of their neuronal functions. In particular, following the deposition of the different nanomaterials on glass substrates, we have evaluated their effects on circuit function and geometry. The geometrical restrictions due to CNT nucleated nodes allowed growth of neuronal networks with control over network geometry, and exploring its functional impact. In diverse applications from basic neuroscience, the presence of CNT nodes may be exploited in brain interfaces able to convey highly localized electrical stimuli.

The Fragile X mental retardation protein (FMRP) is involved in many cellular processes and it regulates synaptic and network development in neurons. Its absence is known to lead to intellectual disability, with a wide range of comorbidities including autism. Over the past decades, FMRP research focused on abnormalities both in glutamatergic and GABAergic signaling, and an altered balance between excitation and inhibition has been hypothesized to underlie the clinical consequences of absence of the protein. Using Fmrp knockout mice, we studied an in vitro model of cortical microcircuitry and observed that the loss of FMRP largely affected the electrophysiological correlates of network development and maturation but caused less alterations in single-cell phenotypes. The loss of FMRP also caused a structural increase in the number of excitatory synaptic terminals. Using a mathematical model, we demonstrated that the combination of an increased excitation and reduced inhibition describes best our experimental observations during the ex vivo formation of the network connections., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the CNS, NMDA receptors generate large and highly regulated Ca 2+ signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca 2+ -dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca 2+ and calmodulin (CaM). We asked whether Ca 2+ influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (κ). In the absence of extracellular Ca 2+ , we observed no cooperativity (κ < 0.1), whereas in 1.8 mm external Ca 2+ , both recombinant and native channels showed substantial negative cooperativity (κ = 0.27). Intracellular Ca 2+ chelation or overexpression of a Ca 2+ -insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially (κ = 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents. SIGNIFICANCE STATEMENT At central synapses, NMDA receptors are a major class of excitatory glutamate-gated channels and a source of activity-dependent Ca 2+ influx. In turn, fluxed Ca 2+ ions bind to calmodulin-primed receptors and reduce further entry, through an autoinhibitory mechanism known as Ca 2+ -dependent inactivation (CDI). Here, we show that the diffusion of fluxed Ca 2+ between active channels situated within submicroscopic distances amplified receptor inactivation. Thus, calmodulin-mediated gating modulation, an evolutionarily conserved regulatory mechanism, endows synapses with sensitivity to both the temporal sequence and spatial distribution of Ca 2+ signals. Perturbations in this mechanism, which coordinates the activity of NMDA receptors within a cluster, may cause signaling alterations that contribute to neuropsychiatric conditions., (Copyright © 2019 the authors.)

Objective: The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, stimulates insulin secretion and efficiently suppresses food intake to reduce body weight. As such, liraglutide is growing in popularity in the treatment of diabetes and chronic weight management. Within the brain, liraglutide has been shown to alter the activity of hypothalamic proopiomelanocortin (POMC) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. Moreover, the acute activities of POMC and NPY neurons have been directly linked to feeding behavior, body weight, and glucose metabolism. Despite the increased usage of liraglutide and other GLP-1 analogues as diabetic and obesity interventions, the cellular mechanisms by which liraglutide alters the activity of metabolically relevant neuronal populations are poorly understood., Methods: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY neurons for patch-clamp electrophysiology experiments., Results: We found that liraglutide directly activated arcuate POMC neurons via TrpC5 channels, sharing a similar mechanistic pathway to the adipose-derived peptide leptin. Liraglutide also indirectly increases excitatory tone to POMC neurons. In contrast, liraglutide inhibited NPY/AgRP neurons through post-synaptic GABA A receptors and enhanced activity of pre-synaptic GABAergic neurons, which required both TrpC5 subunits and K-ATP channels. In support of an additive role of leptin and liraglutide in suppressing food intake, leptin potentiated the acute effects of liraglutide to activate POMC neurons. TrpC5 subunits in POMC neurons were also required for the intact pharmacological effects of liraglutide on food intake and body weight. Thus, the current study adds to recent work from our group and others, which highlight potential mechanisms to amplify the effects of GLP-1 agonists in vivo. Moreover, these data highlight multiple sites of action (both pre- and post-synaptic) for GLP-1 agonists on this circuit., Conclusions: Taken together, our results identify critical molecular mechanisms linking GLP-1 analogues in arcuate POMC and NPY/AgRP neurons with metabolism., (Copyright © 2019. Published by Elsevier GmbH.)

Many efforts have been spent in the last decade for the development of nanoscale synaptic devices integrated into neuromorphic circuits, trying to emulate the behavior of natural synapses. The study of brain properties with the standard approaches based on biocompatible electrodes coupled to conventional electronics, however, presents strong limitations, which in turn could be overcame by the in-situ growth of neuronal networks coupled to memristive devices. To meet this challenging task, here two different chips were designed and fabricated for culturing neuronal cells and sensing their electrophysiological activity. The first chip was designed to be connected to an external memristor, while the second chip was coated with TiO 2 films owning memristive properties. The biocompatibility of chips was preliminary analyzed by culturing the hybrid motor-neuron cell line NSC-34 and by measuring the electrical activity of cells interfacing the chip with a standard patch-clamp setup. Next, neurons were seeded on chips and their activity measured with the same setup. For both cell types total current and voltage responses were evoked and recorded with optimal results with no breakdowns. In addition, an external stimulation was applied to cells through chip electrodes, being effective and causing no damage or pitfalls to the cells. Finally, the whole bio-hybrid system, i.e. the chip interconnected with a commercial memristor, was tested with promising results. Spontaneous electrical activity of neurons grown on the chip was indeed present and this signal was collected and sent to the memristor, changing its state. Taken together, we demonstrated the ability of memristor to work with a synaptic/plastic response together with natural systems, opening the way for the further implementation of basic computing elements able to perform both storage and processing of data, as in natural neurons., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Deficient motivation contributes to numerous psychiatric disorders, including withdrawal from drug use, depression, schizophrenia, and others. Nucleus accumbens (NAc) has been implicated in motivated behavior, but it remains unclear whether motivational drive is linked to discrete neurobiological mechanisms within the NAc. To examine this, we profiled cohorts of Sprague-Dawley rats in a test of motivation to consume sucrose. We found that substantial variability in willingness to exert effort for reward was not associated with operant responding under low-effort conditions or stress levels. Instead, effort-based motivation was mirrored by a divergent NAc shell transcriptome with differential regulation at potassium and dopamine signaling genes. Functionally, motivation was inversely related to excitability of NAc principal neurons. Furthermore, neuronal and behavioral outputs associated with low motivation were linked to faster inactivation of a voltage-gated potassium channel, K v 1.4. These results raise the prospect of targeting K v 1.4 gating in psychiatric conditions associated with motivational dysfunction., Competing Interests: BO, AA, EA, RC, JT, PO No competing interests declared, (© 2019, O'Donovan et al.)

The severity of asthma is closely related to the intensity of airway vagal activity; however, it is unclear how airway vagal activity is centrally augmented in asthma. Here we report that in an asthma model of male Sprague-Dawley rats, the expression and activity of ecto-5'-nucleotidase (CD73) were decreased in airway vagal centers, ATP concentration in cerebral spinal fluid was increased, and the inhibitory and excitatory airway vagal responses to intracisternally injected ATP (5 μmol) and CD73 inhibitor AMPCP (5 μmol), respectively, were attenuated. In airway vagal preganglionic neurons (AVPNs) identified in medullary slices of neonatal Sprague-Dawley rats, AMPCP (100 μmol·L -1 ) caused excitatory effects, as are shown in patch-clamp by depolarization, increased neuronal discharge, and facilitated spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, exogenous ATP (100 μmol·L -1 , 1 mmol·L -1 ) primarily caused inhibitory effects, which are similar to those induced by exogenous adenosine (100 μmol·L -1 ). Adenosine A 1 receptor antagonist CPT (5 μmol·L -1 ) blocked the inhibition of sEPSCs induced by 100 μmol·L -1 exogenous ATP and that by 100 μmol·L -1 exogenous adenosine, whereas 50 μmol·L -1 CPT converted the inhibition of sEPSCs induced by 1 mmol·L -1 ATP to facilitation that was blocked by addition of P2X receptor antagonist PPADS (20 μmol·L -1 ). These results demonstrate that in rat, the sEPSCs of AVPNs are facilitated by extracellular ATP via activation of P2X receptors and inhibited by extracellular adenosine via activation of A 1 receptors; in experimental asthma, decreased CD73 expression and activity in airway vagal centers contribute to the augmentation of airway vagal activity through imbalanced ATP/ADO modulation of AVPNs.

The chemical structures of some antidepressants are similar to those of recently described amine-containing ligands of acid-sensing ion channels (ASICs). ASICs are expressed in brain neurons and participate in numerous CNS functions. As such, they can be related to antidepressant action or side effects. We therefore studied the actions of a series of antidepressants on recombinant ASIC1a and ASIC2a and on native ASICs in rat brain neurons. Most of the tested compounds prevented steady-state ASIC1a desensitization evoked by conditioning acidification to pH 7.1. Amitriptyline also potentiated ASIC1a responses evoked by pH drops from 7.4 to 6.5. We conclude that amitriptyline has a twofold effect: it shifts activation to less acidic values while also shifting steady-state desensitization to more acidic values. Chlorpromazine, desipramine, amitriptyline, fluoxetine, and atomoxetine potentiated ASIC2a response. Tianeptine caused strong inhibition of ASIC2a. Both potentiation and inhibition of ASIC2a were accompanied by the slowdown of desensitization, suggesting distinct mechanisms of action on activation and desensitization. In experiments on native heteromeric ASICs, tianeptine and amitriptyline demonstrated the same modes of action as on ASIC2a although with reduced potency.

Performing electrophysiological recordings from human neurons that have been differentiated in vitro, as compared to primary cultures, raises many challenges. However, patch-clamp recording from neurons derived from stem cells provides an abundance of valuable information, reliably and fast. Here, we describe a protocol that is used successfully in our lab for recording from both control and Fragile X neurons, derived in vitro from human embryonic stem cells.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel expressed in the peripheral and central nervous systems. TRPV1-dependent mechanisms take part in a wide range of physiological and pathophysiological pathways including the regulation of homeostatic functions. TRPV1 expression in the hypothalamus has been described as well as evidence that TRPV1-dependent excitatory inputs to hypothalamic preautonomic neurons are diminished in diabetic conditions. Here we aimed to determine the functional expression of TRPV1 in two hypothalamic nuclei known to be involved in the central control of metabolism and to test the hypothesis that TRPV1-expressing neurons receive TRPV1-expressing inputs. A mouse model (TRPV1 Cre/tdTom ) was generated to identify TRPV1-expressing cells and determine the cellular properties of TRPV1-expressing neurons in adult mice. Our study demonstrated the functional expression of TRPV1 in the dorsomedial hypothalamic nucleus and paraventricular nucleus in adult mice. Our findings revealed that a subset of TRPV1 Cre/tdTom neurons receive TRPV1-expressing excitatory inputs, indicating direct interaction between TRPV1-expressing neurons. In addition, astrocytes likely play a role in the modulation of TRPV1-expressing neurons. In summary, this study identified specific hypothalamic regions where TRPV1 is expressed and functional in adult mice and the existence of direct connections between TRPV1 Cre/tdTom neurons. NEW & NOTEWORTHY Transient receptor potential vanilloid type 1 (TRPV1) is expressed in the hypothalamus, and TRPV1-dependent regulation of preautonomic neurons is decreased in hyperglycemic conditions. Our study demonstrated functional expression of TRPV1 in two hypothalamic nuclei involved in the control of energy homeostasis. Our results also revealed that a subset of TRPV1-expressing neurons receive TRPV1-expressing excitatory inputs. These findings suggest direct interaction between TRPV1-expressing neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a μ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating μ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Objective: Hypothalamic Pro-opiomelanocortin (POMC) and Neuropeptide Y/Agouti-Related Peptide (NPY/AgRP) neurons are critical nodes of a circuit within the brain that sense key metabolic cues as well as regulate metabolism. Importantly, these neurons retain an innate ability to rapidly reorganize synaptic inputs and electrophysiological properties in response to metabolic state. While the cellular properties of these neurons have been investigated in the context of obesity, much less is known about the effects of exercise training., Methods: In order to further investigate this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY/AgRP neurons for patch-clamp electrophysiology experiments., Results: Using whole-cell patch-clamp electrophysiology, we found exercise depolarized and increased firing rate of arcuate POMC neurons. The increased excitability of POMC neurons was concomitant with increased excitatory inputs to these neurons. In agreement with recent work suggesting leptin plays an important role in the synaptic (re)organization of POMC neurons, POMC neurons which express leptin receptors were more sensitive to exercise-induced changes in biophysical properties. Opposite to effects observed in POMC neurons, NPY neurons were shunted toward inhibition following exercise., Conclusions: Together, these data support a rapid reorganization of synaptic inputs and biophysical properties in response to exercise, which may facilitate adaptations to altered energy balance and glucose metabolism., (Copyright © 2018. Published by Elsevier GmbH.)

Histaminergic (HA) neurons located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus fire exclusively during waking and support many physiological functions. We investigated the role of the endovanilloid N-oleoyldopamine (OLDA) in TMN, where dopamine synthesis and its conjugation with oleic acid likely occur. We show that several known targets of OLDA including TRPV1 and cannabinoid receptors are expressed in HA neurons. In contrast to capsaicin, which failed to increase firing of HA neurons in TRPV1 knockout mice (TRPVI KO), OLDA was still able to induce excitation. This excitation was not sensitive to the blockade of cannabinoid receptors 1 and 2 and could result from OLDA interaction with GPR119, as the ligand of GPR119, oleoylethanolamide (OEA), also increased the firing of HA neurons. However, we ruled out this possibility as OEA- (but not OLDA-) excitation was abolished by the PPAR (peroxisome proliferator activated receptor) alpha antagonist MK886. The dopamine uptake blocker nomifensine blanked OLDA-excitation and dopamine receptor antagonists abolished the OLDA action in TRPV1 KO mice. Therefore OLDA excites HA neurons through multiple targets suggesting a central role of the histaminergic system in the behavioral stimulation seen after systemic OLDA application., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Medial prefrontal cortex (mPFC) and amygdala are closely interconnected brain areas that play a key role in cognitive-affective aspects of pain through their reciprocal interactions. Clinical and preclinical evidence suggests that dysfunctions in the mPFC-amygdala circuitry underlie pain-related cognitive-affective deficits. However, synaptic mechanisms of pain-related changes in these long-range pathways are largely unknown. Here we used optogenetics and brain slice physiology to analyze synaptic transmission in different types of amygdala neurons driven by inputs from infralimbic (IL) and prelimbic (PL) subdivisions of the mPFC. We found that IL inputs evoked stronger synaptic inhibition of neurons in the latero-capsular division of the central nucleus (CeLC) of the amygdala than PL inputs, and this inhibition was impaired in an arthritis pain model. Furthermore, inhibition-excitation ratio in basolateral amygdala neurons was increased in the pain model in the IL pathway but not in the PL pathway. These results suggest that IL rather than PL controls CeLC activity, and that changes in this acute pain model occur predominantly in the IL-amygdala pathway.

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

Cellular mechanisms underlying the antinociceptive properties of orexins, a group of neuropeptides produced by the hypothalamus, in the spinal dorsal horn have not been thoroughly investigated. We examined how orexin B affects spontaneous synaptic transmission in lamina II neurons, which play a pivotal role in regulating nociceptive transmission, by applying a whole-cell patch-clamp technique to lamina II neurons in adult rat spinal cord slices. In 66% of neurons tested, bath-applied orexin B concentration dependently produced an inward current at -70 mV and/or increased the frequency of glutamatergic spontaneous excitatory postsynaptic current (sEPSC) without changing its amplitude, in a manner resistant to the voltage-gated Na + -channel blocker tetrodotoxin (TTX). Glycinergic spontaneous inhibitory transmission was enhanced by orexin B in a TTX-sensitive manner in 71% of neurons examined, whereas GABAergic transmission was unaffected in the majority of these neurons. These activities were inhibited by an orexin-2 receptor antagonist (JNJ10397049) but not an orexin-1 receptor antagonist (SB334867). While the effects of orexin B in orexin B-sensitive neurons were mimicked by orexin A, another hypothalamic neuropeptide, oxytocin, produced an inward current but no increase in sEPSC frequency. These results indicate that orexin B produces membrane depolarization and/or increased spontaneous l-glutamate release in lamina II neurons by activating orexin-2 receptors, leading to increased excitability of these neurons. Such increases potentially produce an action potential, resulting in enhancement of glycinergic transmission in lamina II neurons. This activity of orexin B, and possibly orexin A, may contribute to its antinociceptive effects, which are partly shared by oxytocin., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Current strategies in Central Nervous System (CNS) repair focus on the engineering of artificial scaffolds for guiding and promoting neuronal tissue regrowth. Ideally, one should combine such synthetic structures with stem cell therapies, encapsulating progenitor cells and instructing their differentiation and growth. We used developments in the design, synthesis, and characterization of polysaccharide-based bioactive polymeric materials for testing the ideal composite supporting neuronal network growth, synapse formation and stem cell differentiation into neurons and motor neurons. Moreover, we investigated the feasibility of combining these approaches with engineered mesenchymal stem cells able to release neurotrophic factors. We show here that composite bio-constructs made of Chitlac, a Chitosan derivative, favor hippocampal neuronal growth, synapse formation and the differentiation of progenitors into the proper neuronal lineage, that can be improved by local and continuous delivery of neurotrophins., Statement of Significance: In our work, we characterized polysaccharide-based bioactive platforms as biocompatible materials for nerve tissue engineering. We show that Chitlac-thick substrates are able to promote neuronal growth, differentiation, maturation and formation of active synapses. These observations support this new material as a promising candidate for the development of complex bio-constructs promoting central nervous system regeneration. Our novel findings sustain the exploitation of polysaccharide-based scaffolds able to favour neuronal network reconstruction. Our study shows that Chitlac-thick may be an ideal candidate for the design of biomaterial scaffolds enriched with stem cell therapies as an innovative approach for central nervous system repair., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The neuronal population of the subthalamic nucleus (STN) has the ability to prolong incoming cortical excitation. This could result from intra-STN feedback excitation. The combination of inducible genetic fate mapping techniques with in vitro targeted patch-clamp recordings, allowed identifying a new type of STN neurons that possess a highly collateralized intrinsic axon. The time window of birth dates was found to be narrow (E10.5-E14.5) with very few STN neurons born at E10.5 or E14.5. The fate mapped E11.5-12.5 STN neuronal population included 20% of neurons with profuse axonal branching inside the nucleus and a dendritic arbor that differed from that of STN neurons without local axon collaterals. They had intrinsic electrophysiological properties and in particular, the ability to generate plateau potentials, similar to that of STN neurons without local axon collaterals and more generally to that of classically described STN neurons. This suggests that a subpopulation of STN neurons forms a local glutamatergic network, which together with plateau potentials, allow amplification of hyperdirect cortical inputs and synchronization of the STN neuronal population., (© 2017 Wiley Periodicals, Inc.)

Development of functional synapses is crucial for the transmission and storage of information in the brain. Post establishment of the initial synaptic contact, synapses are stabilized through neuronal activity-induced signals. Emerging studies have implicated ubiquitination; a reversible posttranslational modification, as a key regulatory switch that modulates synapse development through proteasomal degradation. Ubiquitination of proteins is precisely regulated by E3 ligases, a set of enzymes that bind to specific substrates to facilitate the conjugation of monomeric or polymeric ubiquitin. However, the identity of specific E3 ubiquitin ligases that influence activity-dependent maturation of synapses and the mechanism by which ubiquitination of proteins regulate functional synapse development remain elusive. Here, we have identified a RING domain containing E3 ligase, Rnf2, asan activity-regulated factor that modulates glutamatergic synapse development in the hippocampus. Rnf2 is a synapse associated E3 ligase that is stabilized by neuronal activity through self-polyubiquitination. We have shown that neuronal activity shifts the balance toward stabilization of Rnf2 through self-polyubiquitination rather than triggering its degradation through polyubiquitination by Ube3A, an E3 ligase implicated in Angelman Syndrome. Our synapse density measurements and whole-cell patch-clamp recordings have revealed that the loss of Rnf2 function in cultured hippocampal neurons result in the development of 'silent' synapses that lack GluA1 containing functional AMPA receptors. These results provide a plausible mechanistic approach toward understanding how synapse maturation is regulated via the activity-dependent stabilization of Rnf2 through a non-canonical function of polyubiquitination., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Preautonomic neurons in the paraventricular nucleus (PVN) of the hypothalamus play a large role in the regulation of hepatic functions via the autonomic nervous system. Activation of hepatic sympathetic nerves increases glucose and lipid metabolism and contributes to the elevated hepatic glucose production observed in the type 2 diabetic condition. This augmented sympathetic output could originate from altered activity of liver-related PVN neurons. Remarkably, despite the importance of the brain-liver pathway, the cellular properties of liver-related neurons are not known. In this study, we provide the first evidence of overall activity of liver-related PVN neurons. Liver-related PVN neurons were identified with a retrograde, trans-synaptic, viral tracer in male lean and db/db mice and whole-cell patch-clamp recordings were conducted. In db/db mice, the majority of liver-related PVN neurons fired spontaneously; whereas, in lean mice the majority of liver-related PVN neurons were silent, indicating that liver-related PVN neurons are more active in db/db mice. Persistent, tonic inhibition was identified in liver-related PVN neurons; although, the magnitude of tonic inhibitory control was not different between lean and db/db mice. In addition, our study revealed that the transient receptor potential vanilloid type 1-dependent increase of excitatory neurotransmission was reduced in liver-related PVN neurons of db/db mice. These findings demonstrate plasticity of liver-related PVN neurons and a shift toward excitation in a diabetic mouse model. Our study suggests altered autonomic circuits at the level of the PVN, which can contribute to autonomic dysfunction and dysregulation of neural control of hepatic functions including glucose metabolism. SIGNIFICANCE STATEMENT A growing body of evidence suggests the importance of the autonomic control in the regulation of hepatic metabolism, which plays a major role in the development and progression of type 2 diabetes mellitus. Despite the importance of the brain-liver pathway, the overall activity of liver-related neurons in control and diabetic conditions is not known. This is a significant gap in knowledge, which prevents developing strategies to improve glucose homeostasis via altering the brain-liver pathway. One of the key findings of our study is the overall shift toward excitation in liver-related hypothalamic neurons in the diabetic condition. This overactivity may be one of the underlying mechanisms of elevated sympathetic activity known in metabolically compromised patients and animal models., (Copyright © 2017 the authors 0270-6474/17/3711140-11$15.00/0.)

Type two voltage gated calcium (Ca V 2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability. 1 Mechanisms that regulate Ca V 2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express Ca V 2.2 and Ca V 2.3. 2 We noted previously that heterologous expression of Ca V 2.1 but not Ca V 2.2 results in increased calcium current in SCG neurons. 3 In the present study, we extended these observations to show that both Ca V 2.1 and Ca V 2.3 expression resulted in increased calcium currents while Ca V 2.2 expression did not. Further, Ca V 2.1 could displace native Ca V 2.2 channels, but Ca V 2.3 expression could not. Heterologous expression of the individual accessory subunits α 2 δ-1, α 2 δ-2, α 2 δ-3, or β4 alone failed to increase current density, suggesting that the calcium current ceiling when Ca V 2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native Ca V 2.2 by recombinant channels. Both α 2 δ-1 and α 2 δ-2 seemed to promote Ca V 2.2 displacement by recombinant channel expression, while α 2 δ-3 appeared to protect Ca V 2.2 from displacement. Thus, we observe a selective prioritization of Ca V channel functional expression in neurons by specific α 2 δ subunits. These data highlight a new function for α 2 δ subtypes that could shed light on subtype selectivity of Ca V 2 membrane expression.

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The basal forebrain (BF) controls sleep-wake cycles, attention and reward processing. Compared to cholinergic and GABAergic neurons, BF glutamatergic neurons are less well understood, due to difficulties in identification. Here, we use vesicular glutamate transporter 2 (vGluT2)-tdTomato mice, expressing a red fluorescent protein (tdTomato) in the major group of BF glutamatergic neurons (vGluT2+) to characterize their intrinsic electrical properties and cholinergic modulation. Whole-cell, patch-clamp recordings were made from vGluT2+ neurons in coronal BF slices. Most BF vGluT2+ neurons were small/medium sized (<20µm), exhibited moderately sized H-currents and had a maximal firing frequency of ∼50Hz. However, vGluT2+ neurons in dorsal BF (ventral pallidum) had larger H-currents and a higher maximal firing rate (83Hz). A subset of BF vGluT2+ neurons exhibited burst/cluster firing. Most vGluT2+ neurons had low-threshold calcium spikes/currents. vGluT2+ neurons located in ventromedial regions of BF (in or adjacent to the horizontal limb of the diagonal band) were strongly hyperpolarized by the cholinergic agonist, carbachol, a finding apparently in conflict with their increased discharge during wakefulness/REM sleep and hypothesized role in wake-promotion. In contrast, most vGluT2+ neurons located in lateral BF (magnocellular preoptic area) or dorsal BF did not respond to carbachol. Our results suggest that BF glutamatergic neurons are heterogeneous and have morphological, electrical and pharmacological properties which distinguish them from BF cholinergic and GABAergic neurons. A subset of vGluT2+ neurons, possibly those neurons which project to reward-related areas such as the habenula, are hyperpolarized by cholinergic inputs, which may cause phasic inhibition during reward-related events., (Copyright © 2017 IBRO. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the brainstem and spinal cord. Clinical studies have indicated that there is a distinct region-dependent difference in the vulnerability of motor neurons. For example, the motor neurons in the facial and hypoglossal nuclei are more susceptible to neuronal death than those in the oculomotor nucleus. To understand the mechanism underlying the differential susceptibility to cell death of the neurons in different motor nuclei, we compared the effects of chemical anoxia on the membrane currents and postsynaptic currents in different motor nuclei. The membrane currents were recorded from neurons in the oculomotor, facial and hypoglossal nuclei in brain slices of juvenile Wistar rats by using whole-cell recording in the presence of tetrodotoxin that prevents action potential-dependent synaptic transmission. NaCN consistently induced an inward current and a significant increase in the frequency of spontaneous synaptic inputs in neurons from these three nuclei. However, this increase in the synaptic input frequency was abolished by strychnine, a glycine receptor antagonist, but not by picrotoxin in neurons from the hypoglossal and facial nuclei, whereas that in neurons from the oculomotor nucleus was abolished by picrotoxin, but not by strychnine. Blocking ionotropic glutamate receptors did not significantly affect the NaCN-induced release facilitation in any of the three motor nuclei. These results suggest that anoxia selectively facilitates glycine release in the hypoglossal and facial nuclei and GABA release in the oculomotor nucleus. The region-dependent differences in the neurotransmitters involved in the anoxia-triggered release facilitation might provide a basis for the selective vulnerability of motor neurons in the neurodegeneration associated with ALS., (Copyright © 2017. Published by Elsevier Inc.)

It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca 2+ -permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca 2+ -impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca 2+ -permeable AMPA receptors (IC 50 =43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The molecular mechanisms underlying acute leptin and serotonin 2C receptor-induced hypophagia remain unclear. Here, we show that neuronal and pro-opiomelanocortin (Pomc)-specific loss of transient receptor potential cation 5 (TrpC5) subunits is sufficient to decrease energy expenditure and increase food intake resulting in elevated body weight. Deficiency of Trpc5 subunits in Pomc neurons is also sufficient to block the anorexigenic effects of leptin and serotonin 2C receptor (Ht2Cr) agonists. The loss of acute anorexigenic effects of these receptors is concomitant with a blunted electrophysiological response to both leptin and Ht2Cr agonists in arcuate Pomc neurons. We also demonstrate that the Ht2Cr agonist lorcaserin-induced improvements in glucose and insulin tolerance are blocked by TrpC5 deficiency in Pomc neurons. Together, our results link TrpC5 subunits in the brain with leptin- and serotonin 2C receptor-dependent changes in neuronal activity, as well as energy balance, feeding behavior, and glucose metabolism., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)